Eur J Biochem, 1983 Nov 15, 136(3), 559 - 70 Mutual conformational changes of tryptophanyl-tRNA synthetase and tRNATrp in the course of their specific interaction; Beresten S et al.; tRNATrp (beef, yeast) is capable of accelerating limited tryptic hydrolysis of the N-terminal part in the polypeptide chains of dimeric beef pancreas tryptophanyl-tRNA synthetase; it can also eliminate the protective effect of tryptophanyl adenylate on the enzyme proteolysis . The effect of tRNA on the proteolysis is manifested even when the 3'-CCA terminus is removed . It has been concluded that the conformation of the synthetase changes when it forms a complex with tRNATrp . Yeast tRNATrp lacking the 3'-half of the acceptor stem can still interact with the synthetase and, to certain extent, induces changes in the conformation of the latter . The susceptibility of single-stranded and double-stranded regions of tRNATrp to cleavage with endonucleases has been studied, and the results are indicative of the fact that, regardless of considerable differences in the nucleotide sequence of yeast and beef tRNATrp, their three-dimensional structures are similar . This fact is consistent with the finding that parameters for the interaction of these tRNAsTrp with beef tryptophanyl-tRNA synthetase are rather close . The three-dimensional structure of tRNATrp is altered when the enzyme forms a complex with it, as seen from (a) a change in the circular dichroic spectrum and (b) an elevated susceptibility of the anticodon and, apparently, acceptor stems to cleavage with nuclease . The conversion of exposed cytidine residues in tRNATrp into uridine residues results in a loss of the acceptor activity; the capability to accelerate limited tryptic hydrolysis of tryptophanyl-tRNA synthetase is also lost although the enzyme-substrate complex, as seen from circular dichroic spectra, can still be formed . The conversion of cytosine in the anticodon stem into uracil modifies the conformation of the anticodon stem . The anticodon arm (including the anticodon) and the acceptor stem play an essential role in the interaction between tRNATrp and tryptophanyl-tRNA synthetase. J Biol Chem, 1983 Nov 10, 258(21), 12768 - 70 The sequence of the nucleotides at the alpha-sarcin cleavage site in rat 28 S ribosomal ribonucleic acid; Chan YL et al.; The sequence of the 521 nucleotides at the 3' end of a rat 28 S rRNA gene was determined . The region encompasses the site of cleavage of 28 S rRNA by the cytotoxin alpha-sarcin . The toxin hydrolyzes a phosphodiester bond on the 3' side of a guanine residue 393 nucleotides from the 3' end . The alpha-sarcin domain is composed of a purine-rich sequence of 14 highly conserved nucleotides. J Biol Chem, 1983 Nov 10, 258(21), 13230 - 5 Cytochrome oxidase subunit 2 gene in Neurospora crassa mitochondria; Macino G et al.; The nucleotide sequence of the cytochrome oxidase subunit 2 (COX2) gene has been obtained from cloned mitochondrial DNA segments of Neurospora crassa . The coding sequences have been identified on the basis of protein sequence homology with the subunit 2 of cytochrome oxidase from yeast and man . The postulated precursor of the N . crassa subunit 2 protein is 250 amino acids long, with a molecular weight of 28,700 . As in the tRNA and rRNA genes, the subunit 2 gene is flanked by G + C-rich palindromic sequences, which are highly conserved in N . crassa mitochondria . Three major transcripts have been detected by Northern blot hybridization . A transcript of 1100 bases is tentatively considered the fully processed mRNA . Furthermore, S1 nuclease protection experiments have revealed that the putative subunit 2 mRNA has a 330 nucleotide long 5' leader sequence. J Microsc, 1983 Nov, 132 (Pt 2), 171 - 8 Bidirectional shadowing in freeze-etching; Willison JH et al.; Bidirectional shadowing in freeze-etching may be achieved by firing an electron-beam shadowing source, rotating the specimen stage through a desired angle, and re-firing the shadowing source . It is demonstrated that portrait shadow-casting, which permits information to be drawn from much of the specimen region lying within primary shadows, can be readily achieved using a 90 degree specimen rotation . With 180 degree specimen rotation, particle-size analysis is feasible . Particle-height analysis is demonstrated using membrane-associated particles as an example . Data from suitable sets of micrographs can also be used for the estimation of particle-width exaggeration due to the accumulation of the shadowing-metal cap . Fibre-width analysis, using the linear regression method, is demonstrated by a study of native cellulosic microfibrils . Mean microfibril widths were found to be 5.5-7.0 nm. Proc Natl Acad Sci U S A, 1983 Nov, 80(22), 6750 - 4 Targeted selection of recombinant clones through gene dosage effects; Rine J et al.; The availability of multicopy plasmid vectors for the yeast Saccharomyces cerevisiae allows the selective amplification of individual segments of the genome . Increased dosage of particular genes results in overproduction of specific gene products and thereby confers resistance to certain metabolic inhibitors . Advantage was taken of this fact to isolate recombinant clones that increase the activities of the enzymes UDP-N-acetylglucosamine-1-P transferase and 3-hydroxy-3-methylglutaryl-CoA reductase. J Gen Microbiol, 1983 Nov, 129 ( Pt 11), 3519 - 23 Studies on the regulation of X-prolyl dipeptidyl aminopeptidase activity; Garcia Ramos C et al.; The specific activity of X-prolyl-dipeptidyl aminopeptidase in Saccharomyces cerevisiae grown on glucose-containing medium remains constant during exponential growth and increases less than twofold when cells reach the stationary phase . In cells harvested from exponential growth on glucose-containing medium the specific activity of the enzyme is found to be 20-30% lower than the specific activity observed in media without glucose, containing acetate or ethanol as the carbon source . X-Prolyl-dipeptidyl aminopeptidase is not inactivated after the addition of glucose to stationary phase cells . Growth of the yeast on poor nitrogen sources or under nitrogen-starvation results in a three- to fourfold increase in the level of the enzyme. Mol Biol Med, 1983 Nov, 1(4), 425 - 45 DNA sequence analysis of the EcoRI Dhet fragment of B95-8 Epstein-Barr virus containing the terminal repeat sequences; Bankier AT et al.; An analysis of the approximately 12,440 base-pair sequence of the EcoRI Dhet fragment isolated from the circular episomal form of the B95-8 strain of Epstein-Barr virus is presented . This fragment contains the covalently joined ends of the intracellular episomal form of the molecule . In the viral capsid the DNA is linear and the joining is mediated via the terminal repeated DNA . Four copies of tandem repeated DNA were present in this clone, three with a repeat size of 538 and one of 523 . The positions of a number of possible protein coding regions and transcription signals are discussed . In particular a possible spliced coding region for an approximately 45,000 Mr protein expressed in latently infected transformed cells is proposed . The predicted protein sequence contains hydrophobic regions separated by charged amino acids reminiscent of a membrane protein. J Immunol Methods, 1983 Oct 28, 63(3), 321 - 7 A human monocyte system for the assay of plasma opsonins; Simpson AW et al.; Previous bioassays for studying the opsonic regulation of the monocyte-macrophage system are subject to a variety of serious disadvantages which limit their value . An assay system is described which avoids many of these pitfalls and is applicable to the rapid assay of large numbers of samples . Human monocytes were isolated from peripheral blood and Saccharomyces cerevisiae used as the target particle . Particle ingestion was assessed using an electronic particle size analyzer and a close correlation was shown between this assay and a visual assessment of ingested micro-organisms. J Biol Chem, 1983 Oct 25, 258(20), 12405 - 8 The catalytic mechanism of transketolase . Thiamin pyrophosphate-derived transition states for transketolase and pyruvate dehydrogenase are not identical; Shreve DS et al.; Thiamin thiazolone pyrophosphate (TTPP) has been reported to be an effective transition state analogue for the thiamin pyrophosphate-dependent partial reaction of pyruvate dehydrogenase (Gutowski, J . A., and Lienhard, G . E . (1976) J . Biol . Chem . 251, 2863-2866) . The kinetics of the interaction of TTPP with transketolase are reported here . TTPP is a competitive inhibitor, with respect to thiamin pyrophosphate, of bakers' yeast transketolase but it is neither a tight binding inhibitor nor a slow binding inhibitor . TTPP decreases the kinetically observed negative cooperativity seen for thiamin pyrophosphate and also decreases the rate constant for the hysteretic activation of the enzyme by thiamin pyrophosphate . We conclude that thiamin thiazolone pyrophosphate is not an effective transition state analogue for the reaction catalyzed by bakers' yeast transketolase . This difference between transketolase and pyruvate dehydrogenase may be related to differences in the polarity of the active sites of the enzymes . It is conceivable that the active sites of the pyruvate decarboxylase subunit of pyruvate dehydrogenase is hydrophobic, by analogy with the known hydrophobicity of the active site of brewers' yeast pyruvate decarboxylase . This hydrophobicity would stabilize a transition state with no charge on the thiazole portion of the coenzyme, similar to the "uncharged" thiazole portion of TTPP . In contrast, the active site of bakers' yeast transketolase, which is known to contain charged amino acid side chains, should be less favorable for such an uncharged transition state . A charge-separated canonical form related to TTPP could be preferentially stabilized in the active site of transketolase. J Biol Chem, 1983 Oct 25, 258(20), 12132 - 4 Fluorescence decay time measurements of Eu3+-ATP-enzyme complexes . Replacement of the metal hydration water by active site ligands; Gutman M et al.; Measurements of the fluorescent lifetimes of the rare earth metal Eu3+ in varying mole fractions of H2O/D2O were used to determine the hydration of the metal in the presence of ATP and/or hexokinase or chloroplast reversible ATPase . The number of water molecules coordinated to the metal in Eu3+-ATP was estimated to be 2.6; when this complex is bound to hexokinase, 1 water molecule is displaced . Upon binding to chloroplast reversible ATPase, the metal coordinates 1 water molecule while the Eu3+-ATP complex does not retain any associated solvent . These numbers are in contrast to the 9 solvent molecules coordinated to the naked metal ion . These results are discussed in reference to mechanistic and structural considerations of the two enzymes. FEBS Lett, 1983 Oct 17, 162(2), 300 - 4 Evidence that the multifunctional polypeptides of vertebrate and fungal fatty acid synthases have arisen by independent gene fusion events; McCarthy AD et al.; The enoyl reductase (NADPH binding site) of rabbit mammary fatty acid synthase has been radioactively labelled using pyridoxal phosphate and sodium {3H}borohydride . Using this method we have been able to add this site to the four sites whose location has already been mapped within the multifunctional polypeptide chain of the protein . The results show that the enoyl reductase lies between the 3-oxoacylsynthase and the acyl carrier . This confirms that the active sites occur in a different order on the single multifunctional polypeptide of vertebrate fatty acid synthase and the two multifunctional polypeptides of fungal fatty acid synthase, and suggests that these two systems have arisen by independent gene fusion events. J Biomol Struct Dyn, 1983 Oct, 1(2), 357 - 69 Molecular dynamics of phenylalanine transfer RNA; Prabhakaran M et al.; The atomic motions of yeast phenylalanine transfer RNA have been simulated using the molecular dynamics algorithm . Two simulations were carried out for a period of 12 picoseconds, one with a normal Van der Waals potential and the other with a modified Van der Waals potential intended to mimic the effect of solvent . An analysis of large scale motions, surface exposure, root mean square displacements, helical oscillations and relaxation mechanisms reveals the maintenance of stability in the simulated structures and the general similarity of the various dynamic features of the two simulations . The regions of conformational flexibility and rigidity for tRNA(Phe) have been shown in a quantitative measure through this approach. J Biomol Struct Dyn, 1983 Oct, 1(2), 337 - 55 Loop stereochemistry and dynamics in transfer RNA; Westhof E et al.; The stereochemistry and the dynamics of two loops of yeast tRNA-asp, the thymine loop and the anticodon loop, are compared in the hope of a better understanding of the relationships between loop sequence and loop topology . Both loops are seven residues long and both present sharp turns after the second residue, U33 and psi 55, stabilized by hydrogen bonds between N3-H of the pyrimidine and the phosphates of C36 and A58 and stacking interactions of the pyrimidine ring with the phosphates of U35 and A57, respectively . In the thymine loop, the two purines following C56, A57 and A58, open up to leave space for the intercalation of the first invariant guanine residue of the D-loop, while the two pyrimidine bases, which follow A58, turn away from the stacking pattern of the thymine arm and stack instead with the last base pair of the dihydrouridine arm A15-U48 . In the anticodon loop, however, the bases G34 to C38 form an helical stack in continuity with the anticodon stem on the 3'-end . At the same time C36 forms Watson-Crick hydrogen bonds with G34 of a twofold symmetrically related molecule . The anticodon-anticodon base pairing interactions between symmetrically-related molecules are stabilized by stacking with the modified base G37 on both sides of the triplet . Some comparisons are made with the structure of yeast tRNA-phe and some implications about the structure of mitochondrial tRNAs are discussed. Cryobiology, 1983 Oct, 20(5), 542 - 52 Osmotic response of individual cells during freezing . II . Membrane permeability analysis; Schwartz GJ et al.; An analytical model is presented to simulate the freezing of individual yeast cells . In addition the model is solved numerically on a digital computer to obtain values for cell volume as a function of temperature, based on the thermal protocol during freezing, and the transport parameters of the cell membrane . The numerical procedure was modified to enable values for the membrane hydraulic permeability reference coefficient, Lpg, and activation energy, ELp, to be deduced by nonlinear analysis of complementary experimental data (10) . It was observed that the apparent values of both Lpg and ELp increase with cooling rate, from Lpg = 0.0116 micrometer 3 micrometers-2 atm-1 min-1 and ELp = 19.4 kJ mol-1 for 9 degrees K/min to Lpg = 2.11 micrometers 3 micrometer-2 atm-1 min-1 and ELp = 101 kJ mol-1 for 35 degrees K/min . The deduced permeabilities fall within the range of values determined in a prior study by Levin (6) . Analysis with the model also indicates that the turgor pressure exerts a negligible effect on yeast exposed to freezing stress. Br J Dermatol, 1983 Oct, 109(4), 421 - 7 Photobiological activity of suction blister fluid from patients treated with 8-methoxypsoralen; Dubertret L et al.; Suction blister fluid was collected from normal human volunteers before (SBF) and 2 h after (SBF 8-MOP) oral 8-methoxypsoralen (0.6 mg/kg) ingestion without irradiation . In SBF 8-MOP the concentration of 8-MOP was 150 ng/ml, and fluorescent metabolites were also present . The toxicity of SBF 8-MOP was then determined with and without UV-A irradiation in the diploid strain D7 of the yeast Saccharomyces cerevisiae which is suitable for the detection of lethal, mutagenic and recombinogenic events . It was compared with that of SBF, SBF with 8-MOP added in vitro (150 ng/ml) and 8-MOP (150 ng/ml) in water . SBF showed no effect with UV-A doses up to 360 kJ m-2; SBF 8-MOP showed a slight decrease in survival and a dose-dependent increase in nuclear mutations, mitotic gene conversion and crossing over; SBF with 8-MOP added in vitro showed increased photobiological activity compared with SBF 8-MOP . This photobiological activity was increased by a factor of six when using 8-MOP in water . These results indicate a different bioavailability of 8-MOP in water and in interstitial fluid containing proteins . The metabolites of 8-MOP in human skin did not increase the photobiological activity of the drug . We conclude that the 8-MOP present in human skin during PUVA therapy is mutagenic and recombinogenic for eukaryotic cells. Mol Cell Biol, 1983 Oct, 3(10), 1886 - 7 Empirical equation that can be used to determine genetic map distances from tetrad data; Ma C et al.; An empirical equation has been developed that can be used to calculate genetic map distances from tetrad data with good accuracy for distances of up to at least 120 centimorgans. Proc Natl Acad Sci U S A, 1983 Oct, 80(19), 5857 - 61 Type I and type II keratins have evolved from lower eukaryotes to form the epidermal intermediate filaments in mammalian skin; Fuchs E et al.; We have traced the evolutionary origins of keratin-like sequences to the genomes of lower eukaryotes . The proteins encoded by these genes have evolved to form the intermediate filaments that comprise the backbone of vertebrate skin cells . Two related but distinct types of keratins encoded by two separate multigene subfamilies are expressed in the epidermal keratinocytes of vertebrate species from fish to human . Both at the level of protein and at the level of DNA, these two classes of keratins are coordinately conserved throughout vertebrate evolution, indicating the central role that both types of keratins must play in the assembly and structure of the 8-nm filament. Biokhimiia, 1983 Oct, 48(10), 1643 - 53 {Two-stage mechanism of the fluoride inhibition of inorganic pyrophosphatase using the fluoride ion}; Smirnova IN et al.; Some kinetic and spectral approaches have been used to study the interactions in the enzyme-Mg2+-F--pyrophosphate (or imidodiphosphate, a non-hydrolyzeable pyrophosphate analog) system underlying the mechanism of yeast inorganic pyrophosphatase inhibition by fluoride . The continuous curves of the enzymatic reaction were obtained with an automatic phosphate analyzer operating on the time scale of seconds . Increasing concentrations of NaF caused an increase in the inactivation rate constant to a constant level of 5.3 min-1 for PPi (pH 6.2-7.2) and 3.9 min-1 for imidodiphosphate, (pH 7.2) . At a saturating fluoride concentration, the initial rate of PPi hydrolysis dropped to 10% . NaF and imidodiphosphate changed the protein spectrum at 270-310 nm and strengthened the binding of each other to the protein . The binding of F- required a Mg2+-binding site with Kd = 0.15 mM being filled in . The free enzyme and its Ca2+ complex did not bind F- . The experimental results indicate that pyrophosphatase inhibition by fluoride occurs in two steps . The inhibitor adds first to the Mg2+ ion on the enzyme in a readily reversible reaction causing a 90% decrease of the catalytic activity . Thereafter, a slow isomerization of the enzymesubstrate complex takes place, resulting in a complete loss of activity. Biochemistry, 1983 Sep 27, 22(20), 4637 - 41 Kinetic mechanism of the reaction catalyzed by nuclear histone acetyltransferase from calf thymus; Wong LJ et al.; The kinetic mechanism for calf thymus histone acetyltransferase A has been determined from the initial velocity studies . The kinetic patterns at low substrate concentrations suggest that the reaction proceeds via two half-reactions as in a ping-pong pathway with the formation of an acetyl-enzyme intermediate . Such acetyl-enzyme has been isolated and found to be chemically competent . In addition, product inhibition patterns by coenzyme A are consistent with a hybrid ping-pong mechanism . These findings indicate that the acetyltransferase A from calf thymus has two separate and independent binding sites, one for each of the two substrates . Consequently, the mechanism constructed for the acetyltransferase A catalyzed reaction may be described as a double-displacement, two-site ping-pong mechanism. J Biol Chem, 1983 Sep 25, 258(18), 10963 - 6 Evidence for formation of two thioether bonds to link heme to apocytochrome c by partially purified cytochrome c synthetase; Taniuchi H et al.; Cytochrome c synthetase has been solubilized from yeast mitochondria using Triton X-100 and fractionated with ammonium sulfate . Use of this partially purified enzyme has permitted us to isolate a quantity of iso-1-cytochrome c formed from 125I-labeled apocytochrome c and hemin in the presence of a NADPH-generating system . Visible absorption spectra (pH 8.0 or 5.0) including alpha, beta, and Soret bands and their molar absorption coefficients of this enzymatically synthesized cytochrome c in the oxidized and reduced states are the same, within experimental error, as those of native cytochrome c . Pyridine ferrohemochrome (pH 13) of the synthesized species also exhibits the same alpha and beta bands as those of iso-l-cytochrome c and similar to those reported for heme peptides of cytochrome c . If only one or no thioether bond were formed between the two vinyl side groups of heme and the cysteine residues of apocytochrome c, all these alpha and beta bands would have shifted to red (Pettigrew, G . W., Leaver, J . L., Meyer, T . E., and Ryle, T . E . (1975) Biochem J . 147, 291-302) . Thus, two thioether bonds appear to be formed to link heme to apocytochrome c by cytochrome c synthetase, completing information of the three-dimensional structure of cytochrome c. Nucleic Acids Res, 1983 Sep 24, 11(18), 6571 - 86 Similar binding of the carcinostatic drugs cis-{Pt(NH3)2Cl2} and {Ru(NH3)5Cl} Cl2 to tRNAphe and a comparison with the binding of the inactive trans-{Pt(NH3)2Cl2} complex - reluctance in binding to Watson-Crick base pairs within double helix; Rubin JR et al.; A comparative study of the binding of square planar cis- and trans-{Pt(NH3)2Cl2} complexes and the octahedral {Ru(NH3)5(H2O)}3+ complex to tRNAphe from yeast was carried out by X-ray crystallography . Both of the carcinostatic compounds, cis-{Pt(NH3)2Cl2} and {Ru(NH3)5(H2O)}3+ show similarities in their mode of binding to tRNA . These complexes bind specifically to the N(7) positions of guanines G15 and G18 in the dihydrouridine loop . {Ru(NH3)5(H2O)}3+ has an additional binding site at N(7) of residue G1 after extensive soaking times (58 days) . A noncovalent binding site for ruthenium is also observed in the deep groove of the acceptor stem helix with shorter (25 days) soaking time . The major binding site for the inactive trans-{Pt(NH3)Cl2} complex is at the N(1) position of residue A73, with minor trans-Pt binding sites at the N(7) positions of residues Gm34, G18 and G43 . The similarities in the binding modes of cis-{Pt(NH3)2Cl2} and {Ru(NH3)5(H2O)}3+ are expected to be related to their carcinostatic properties. Biochemistry, 1983 Sep 13, 22(19), 4380 - 8 Structure of phenylalanine-accepting transfer ribonucleic acid and of its environment in aqueous solvents with different salts; Li ZQ et al.; Thermodynamic and structural parameters were measured for brewers' yeast tRNAPhe in solution in the range of 0.1-0.9 M monovalent salt (with and without 1 mM MgCl2), pH 7.0, by small-angle neutron scattering . Partial specific volumes and preferential interaction parameters were found to be similar to corresponding values measured by more conventional means in DNA {Eisenberg, H . (1981) Q . Rev . Biophys . 14, 141-172} . There is no evidence of a large conformational change in tRNAPhe in this range, and the molecule has a radius of gyration that is the same as that calculated from the crystal-structure coordinates (23 A) . Transfer RNA in solution is made up of polyion tRNA76- and 76 positive monovalent ions (in absence of Mg2+) . The data show the polyion to be surrounded by a shell of solvent that is significantly denser than bulk, whose structure depends on salt conditions . In 0.1 M NaCl, it has an excess mass of approximately 85 molecules of water . This would be accounted for, for example, by approximately 850 molecules of water if their density were 10% higher than that for bulk . The radius of gyration of the dense shell is approximately 30 A for NatRNA and approximately 35 A for KtRNA . The present study shows that the solvent around tRNA is a component of its structure that must be taken into account in understanding its function. Tohoku J Exp Med, 1983 Sep, 141(1), 33 - 9 Effects of pantethine and its metabolites on fatty acid oxidation in rat liver mitochondria; Morisaki N et al.; The mechanism of the activating effect of pantethine {D-bis-(N-pantothenyl-beta-aminoethyl)disulfide} on fatty acid oxidation was investigated in rat liver mitochondria . Pantethine, pantetheine and 4'-phosphopantetheine activated three steps of fatty acid oxidation, i.e., acyl-CoA synthetase, carnitine, acyltransferase and intramitochondrial oxidation, to various extents . Although their effects may have been partly due to CoASH derived from them, they also had specific effects. Mutat Res, 1983 Sep, 123(1), 31 - 46 An evaluation of the mutagenic, carcinogenic and teratogenic potential of microwaves; Leonard A et al.; A notable proportion of the population is exposed to an increasing number of devices emitting microwaves, a form of non-ionizing electromagnetic radiation in the range 300-30000 mHz . The activation energy of microwave radiations is too small to directly modify any chemical bonds in the irradiated matter . At microwave frequencies the macroscopic dielectric properties of tissues are strongly determined by their water content . Tissues like muscle, brain, skin, with a high water content, have higher permittivity and conductivity values than bone or fat with low water contents . Owing to the energy transfer, to living tissues, by a dipolar relaxation mechanism of water molecules, the penetration of microwaves is limited and one observes a fast and very efficient heat-loss production . A review of the available literature shows that most results on the mutagenicity of microwaves are negative or can often be explained by a temperature enhancement . If microwaves are apparently unable to damage DNA at sub-thermal exposure levels, some results indicate, however, that they might easily potentiate the damaging action of other DNA antagonist agents such as UV or chemicals. Anal Biochem, 1983 Sep, 133(2), 409 - 16 Affinity precipitation of dehydrogenases; Flygare S et al.; Affinity precipitation, a novel technique closely related to immunoprecipitation and affinity chromatography, has been evaluated in systems comprised of dehydrogenases and a bifunctional NAD derivative, Bis-NAD . Lactate dehydrogenase and glutamate dehydrogenase were easily precipitated whereas yeast alcohol dehydrogenase required the presence of salt to enhance the affinity precipitation . Liver alcohol dehydrogenase did not precipitate, probably because most of the affinity complexes formed were composed of only two enzyme molecules . Affinity precipitation was carried out on a preparative scale for the isolation of ox heart lactate dehydrogenase from a crude extract . The yield and purity of the enzyme and the general properties of the procedure are considered very satisfactory. Proc Natl Acad Sci U S A, 1983 Sep, 80(18), 5675 - 9 Recombination between sequences in nonhomologous positions; Sugawara N et al.; Crossing-over between the dispersed repeated sequences found in eukaryotic genomes would generate chromosomal rearrangements . The stability of the yeast genome suggests the existence of some constraint on the ability of these sequences to interact by recombination . We have constructed strains with two alleles of the his3 gene located on different chromosomes . Gene conversion accounts for the majority of the recombination events between these genes, but about 10% of the events are crossovers that result in a reciprocal translocation . When one of the alleles is on an autonomously replicating centromere plasmid, recombination is 5- to 10-fold more frequent than when both alleles are chromosomal, suggesting that higher-order chromosome structure may play a role in restricting interchromosomal recombination . We have also used the translocation to deduce the orientation of the his3 and rRNA genes relative to their centromeres. Biochemistry, 1983 Aug 30, 22(18), 4238 - 47 Examination of the solvent perturbation technique as a method to identify enzyme catalytic groups; Grace S et al.; The present study was undertaken for the purpose of evaluating the solvent perturbation technique as a method to identify enzyme catalytic residues . For establishment of expected directions and sizes of pKa perturbations for different types of acids in different classes of solvents, a study of the pKa of a series of acids in mixed solvent systems was carried out . Consistent with previous findings, the presence of organic solvents (25% v/v) increased the pKa values of neutral acids while it decreased or did not change the pKa values of cationic acids . The size of the perturbation observed was dependent on the nature of the organic solvent and on the polarity of the neutral form of the acid . The solvent perturbation studies were then extended to the catalytic aspartate residue of yeast hexokinase . The pKa of this residue was determined from the MgATP V/K profile measured in the presence and absence of organic solvents (25% v/v) . While dimethylformamide and methanol induced small but perhaps significant increases in the observed pKa, dimethyl sulfoxide and propylene glycol did not . The pKa values, from the MgATP V/K profiles measured in the presence of fully saturating glucose, were not significantly increased by the organic solvents . The pKi vs . pH profile for the competitive inhibitor lyxose was also measured in the presence and absence of organic solvents . While methanol (25% v/v), dimethylformamide (25% v/v), and dioxane (17.5% v/v) induced a large increase in the pKa, propylene glycol and dimethyl sulfoxide (25% v/v) did not . The results from this investigation indicate that the solvent perturbation technique should not be relied upon indiscriminately. Nucleic Acids Res, 1983 Aug 25, 11(16), 5347 - 60 The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences; Woudt LP et al.; Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X . laevis as hybridization probes . A 2.6 kb HindIII-generated N . crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector . Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4 . Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N . crassa . The two genes are physically linked on the genome . DNA sequencing of the N . crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene . The amino acid sequences of the N . crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions . Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes. J Biol Chem, 1983 Aug 25, 258(16), 10049 - 53 The effect of RNA secondary structure on the action of a nucleolar endoribonuclease; Eichler DC et al.; The effect of the folded macromolecular structure of RNA on the action of a purified single strand specific nucleolar ribonuclease was studied by comparing the limited hydrolysis of defined RNA substrates . The nucleolar RNase was shown to attack only single-stranded regions of the native 5.8 S rRNA, consistent with a computer-derived model for the secondary structure (Nazar, R . N., Stitz, T . O., and Busch, H . (1975) J . Biol . Chem . 250, 8591-8597) . The single strand specific nucleolar RNase, unlike S1 nuclease, does not release the end-labeled nucleotide and therefore provides a more useful probe for structural analysis at or near 3'- or 5'-terminal ends of an RNA molecule . Although attacking only single strand regions of the native 5.8 S rRNA, the selectivity of the nucleolar RNase, when compared to S1 nuclease, is distinct and supports the suggestion that other factors besides the proposed secondary structure must also influence nuclease attack . The selective and limited attack of the nucleolar RNase on native RNA was similarly observed using mouse 45 S preribosomal RNA as a substrate and possibly suggests a role for this nucleolar RNase in ribosomal RNA processing. Biochim Biophys Acta, 1983 Aug 1, 752(3), 474 - 81 Acyl-CoA synthetase activity of rat heart mitochondria . Substrate specificity with special reference to very-long-chain and isomeric fatty acids; Normann PT et al.; The acyl-CoA synthetase (acid: CoA ligase (AMP-forming), EC 6.2.1.3) activity of rat heart has been measured in fatty acid-depleted fractions of mitochondria, microperoxisomes and microsomes . The assay was based on (i) the measurement of the reaction product AMP by high-performance liquid chromatography or (ii) a coupled reaction in which the intramitochondrial (matrix) CoASH is the final acyl acceptor and the redox state of the flavoproteins in the acyl-CoA dehydrogenase pathway is used to determine the intramitochondrial level of acyl-CoA . This spectrophotometric method was also used to estimate the 'outer' carnitine long-chain acyltransferase (palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) activity . Comparison of the distribution of long-chain acyl-CoA synthetase activity and marker enzymes in the various subcellular fractions revealed that the synthetase activity is exclusively localized in the mitochondrial fraction . Experimental evidence is presented in support of the conclusion that the chain-length specificity of saturated and monounsaturated fatty acids (16:1-22:1) for the acyl-CoA synthetase is mainly determined by the availability of the fatty acid at the active site, which is largely determined by the affinity of binding of fatty acids to the bulk phase of the mitochondrial phospholipids . Among the 22:1 isomers, 22:1(11) (cis) (cetoleic acid) revealed a slightly higher activity (1.4-fold) than 22:1(13) (cis) (erucic acid) . The polyunsaturated fatty acids tested were rather poor substrates . Using isolated intact mitochondria and 16:0 or 22:1(13) (cis) as the substrates, it was found that the initial rate of the 'outer' long-chain acyltransferase activity was approximately four times higher than that of the long-chain acyl-CoA synthetase . The data support the hypothesis that the long-chain acyl-CoA synthetase reaction is rate-limiting in the sequence of coupled reactions leading to beta-oxidation in the mitochondrial matrix. Arch Biochem Biophys, 1983 Aug, 225(1), 157 - 63 Nitro analogs of substrates for adenylosuccinate synthetase and adenylosuccinate lyase; Porter DJ et al.; The reactivities of the nitro analogs of the substrates of adenylosuccinate synthetase and adenylosuccinate lyase, the enzymes which catalyze the penultimate and last step, respectively, in the pathway for AMP biosynthesis have been examined . Alanine-3-nitronate, an aspartate analog, was a substrate for the synthetase from Azotobacter vinelandii, having a kcat/Km which was approximately 30% that for aspartate . The product of this reaction was N6-(L-1-carboxy-2-nitroethyl)-AMP . Of nine other substrate analogs tested, only cysteine sulfinate (having 5.5% of the activity of aspartate) was reactive . These results demonstrate the strict requirement of the synthetase for a negatively charged substituent, with a carboxylate-like geometry, at the beta-carbon of the alpha-amino acid substrate . The lyase, purified to homogeneity from brewer's yeast by a new procedure, did not utilize N6-(L-1-carboxy-2-nitroethyl)-AMP as a substrate . However, the nitronate form of this analog was a good inhibitor of the lyase (Km/Ki = 28 when compared to adenylosuccinate), suggesting that it mimics a carbanionic intermediate in the reaction pathway . The avid binding of bromphenol blue by the lyase (Ki = 0.95 microM) was used for active site titrations and for displacement of the enzyme, in the purification protocol, from blue Sepharose. Arch Biochem Biophys, 1983 Aug, 225(1), 110 - 5 omega-haloalkyl esters of 5'-adenosine monophosphate as potential active-site-directed reagents for dehydrogenases; Fries RW et al.; A series of esters of adenosine 5'-monophosphate with ethyl, propyl, or hexyl moieties substituted at the omega-position with chlorine or bromine were prepared . The compounds were competitive inhibitors of horse liver alcohol dehydrogenase with respect to coenzyme, NAD+, and had inhibition (dissociation) constants in the range of 40 to 260 microM at pH 8.0, 25 degrees C . The bromoalkyl esters were designed to be active-site-directed inactivators and were chemically reactive as tested with the model compound 4-(p-nitrobenzyl)pyridine . Yeast alcohol dehydrogenase was inactivated by the bromohexyl analog by an active-site-directed mechanism, with a Ki = 1.5 mM and a pseudo-bimolecular rate constant of 0.03 M-1 S-1, which is 150 times larger than the bimolecular rate constant for inactivation by 2-bromoethanol . However, the rates of inactivation of other dehydrogenases treated with 10 mM concentrations of these compounds were generally slower than with the simpler reagent, 2-bromoethanol . Thus, the reactive functional group attached to the AMP moiety may not be properly oriented for affinity labeling of these dehydrogenases . The bromoalkyl esters may be useful for inactivating other enzymes. J Cell Biol, 1983 Aug, 97(2), 317 - 22 Acanthamoeba discriminates internally between digestible and indigestible particles; Bowers B et al.; The capacity of Acanthamoeba to distinguish nutritive yeast particles from non-nutritive plastic beads during phagocytosis was investigated . When cells were allowed to phagocytose yeast to capacity, endocytosis stopped and subsequent presentation of particles (either yeast or beads) did not result in further uptake . By contrast, when cells were allowed to phagocytose plastic beads to capacity and a second dose of particles was presented (either yeast or beads), the cells exocytosed the internal particles and took up new ones . Yeast rendered indigestible by extensive chemical cross-linking were taken up at rates similar to those of untreated yeast, but, like beads, they were exocytosed when a second dose of particles was presented . The results show that an internal distinction is made between vacuoles containing yeast and vacuoles containing plastic beads, and they are consistent with the hypothesis that the presence within the vacuoles of material capable of being digested prevents exocytosis. Endocrinology, 1983 Aug, 113(2), 604 - 10 Brown adipose tissue metabolism in streptozotocin-diabetic rats; Seydoux J et al.; Defects of both diet-induced thermogenesis and cold tolerance have been reported for streptozotocin-diabetic rats . Since brown adipose tissue (BAT) is a major effector of both diet- and cold-induced thermogenesis in the rat, the possible cause of these defects was investigated by comparing BAT metabolism under basal conditions and during activation by nerve stimulation, norepinephrine (NE), or octanoate addition in both streptozotocin-diabetic rats and in controls . The following metabolic indices were measured in rat interscapular BAT (IBAT): 1) tissue composition, 2) heat production rate as measured by direct microcalorimetry, 3) redox state of flavoproteins linked to the acyl-coenzyme A dehydrogenase pathway as measured by reflection spectrometry, 4) redox state of NAD(P) as measured by surface-emitted fluorescence, and 5) fatty acid activation and beta-oxidation activities in IBAT homogenate . In streptozotocin-diabetic rats, IBAT was atrophied (DNA content unmodified, protein and lipid content decreased) . The basal and NE-stimulated total heat production rates showed a 75% and 56% decrease, respectively . The specific activity of fatty acid beta-oxidation as measured by flavoprotein redox state or enzymatically was decreased by 52% and 59%, respectively . The basal redox level of NAD(P) was about 3 times higher than in the controls and NE stimulation resulted in oxidation in contrast to the reduction observed in control tissues . These results show that the metabolic capacity of IBAT from streptozotocin-diabetic rats is decreased and further suggest that the reduced capacity for beta-oxidation contributes significantly to the metabolic alteration. FEBS Lett, 1983 Jul 25, 158(2), 343 - 8 Probing eukaryotic RNA polymerases B with monoclonal antibodies; Vilamitjana J et al.; Monoclonal antibodies directed against RNA polymerase B of the fungus Podospora comata were selected on the basis of different subunits recognition and inhibitory effect on enzyme activity . A library of 10 antibodies biased toward B180, B145, B39, B23,5 and B11 subunits was constructed . Most of these antibodies also recognize yeast, wheat germ and calf thymus RNA polymerase B . Subunits bearing antigenic determinants are not always homologous in Podospora and yeast enzyme . As some of these antibodies strongly inhibit enzyme activity they constitute potent probes for functional studies of corresponding subunits. Nature, 1983 Jul 21-27, 304(5923), 234 - 41 Drosophila melanogaster mitochondrial DNA, a novel organization and genetic code; de Bruijn MH; The sequence of a 4,869 base-pair fragment of Drosophila melanogaster mitochondrial DNA is presented . It contains genes for cytochrome oxidase subunits I, II and III, ATPase subunit 6 and six tRNAs together with two unassigned reading frames . The gene organization differs from that of mammalian mitochondrial DNAs . Evidence is provided for a genetic code in which AGA codes for serine and the quadruplet ATAA is used in initiation of translation. Arch Biochem Biophys, 1983 Jul 15, 224(2), 416 - 28 Intracellular diffusion of water; Tanner JE; Self-diffusion of cell water has been measured at diffusion times ranging from 0.3 ms to 1.0 s for human red cells, yeast, and brine shrimp using various pulsed gradient NMR methods . Intracellular diffusion coefficients and membrane permeabilities are calculated from these data with the aid of previous theoretical results for regularly spaced permeable planar barriers . The intracellular diffusion coefficients of water range from 1.2 X 10(-6) to 6 X 10(-6) cm2/s for the various samples . Outer-membrane permeabilities to water range from 0.0001 to 0.01 cm/s . The self-diffusion coefficient of lipid in a sample of human breast adipose tissue was found to be 1.5 X 10(-7) cm2/s. FEBS Lett, 1983 Jul 11, 158(1), 53 - 7 Dissociation of nucleoprotein complexes by chaotropic salts; Damodaran S et al.; The effect of various anions in destabilizing yeast nucleoprotein complexes followed the order F- less than Cl- less than Br- less than ClO-4 congruent to Cl3CCOO- . Treatment of yeast nucleoproteins with 0.5 M NaClO4 resulted in removal of 80% of RNA . Based on the results, a simple method for effective separation of RNA from ribosomal particles is proposed and the mechanism of RNA dissociation by anions is also discussed. J Biol Chem, 1983 Jul 10, 258(13), 8429 - 35 Crystallization of a tRNA . aminoacyl-tRNA synthetase complex . Characterization and first crystallographic data; Lorber B et al.; A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant . The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry . Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule . It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio . The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432 . The cell parameter is 354 A and the asymmetric unit contains one particle of complex . The solvent content is about 78%, higher than the values commonly observed . Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution. FEBS Lett, 1983 Jul 4, 157(2), 233 - 9 The oxygen binding site of cytochrome oxidase . Structural predictions on subunit I from amino acid sequences; Welinder KG et al.; Analyses of heme-attached amino acid sequences in known hemoprotein superfamilies provide a basis for prediction of such sequences in hemoproteins of unknown three-dimensional structure . Among 11 histidine residues conserved in subunit I of 3 mammalian and 2 fungal cytochrome oxidases the sequence around His-233 (human) is the most conserved and shows remarkable similarity to the sequence of the oxygen binding site in globins . Furthermore, the gene coding for subunit I in Saccharomyces cerevisiae and the gene for leghemoglobin in soybean are both split by introns right after these similar histidine sequences . The predicted distal histidine sequence of subunit I provides for heme a3 and Cua3 binding and has an extraordinarily high content of aromatic residues . These aromatic groups may serve as a molecular electron capacitor . Transmembrane sequences and electron transfer sequences are proposed. Mikrobiologiia, 1983 Jul-Aug, 52(4), 586 - 90 {1,3-beta-Glucanases of actinomycetes}; Tiunova NA et al.; Different actinomyces species (25 strains) were studied and Actinomyces cellulosae 41 was found to be the most active one in its capacity to cause lysis of Saccharomyces cerevisiae intact cells as well as in the production of 1,3-beta-glucanase . The enzyme preparation containing 1,3-beta-glucanase can be obtained from the filtrate of the actinomycete cultural broth after 60 h of growth by precipitation with four volumes of ethanol . The optimal pH for the action of 1,3-beta-glucanase from A . cellulosae is 5.5 . Hydrolysis of laminarin (5 mg of the substrate) yields 2.4 mg of reducing sugars or 13.3 microM (recalculated per glucose) . There is a direct correlation between the amount of produced reducing sugars and the enzyme concentration up to 10 microM/ml (recalculated per glucose) . The enzyme hydrolysate contains glucose and oligosaccharides with a different degree of polymerization . Therefore, the enzyme is endo-1,3-beta-glucanase . It produces less quantities of the disaccharide than those of glucose and trisaccharide . The enzyme yields only traces of glucose upon prolonged hydrolysis of p-nitrophenyl-beta-D-glucoside (PNPG) and shows a weak capacity for transglycosylation when laminarin is used as a donor and PNPG as an acceptor. Rev Infect Dis, 1983 Jul-Aug, 5 Suppl 3, S614 - 9 Antifungal action of rifampin; Medoff G; Rifampin and amphotericin B were found to be synergistic in vitro against Saccharomyces cerevisiae, Histoplasma capsulatum, and several species of Aspergillus, The uptake of rifampin by the fungi was increased in the presence of amphotericin B, and this increase probably formed the basis for the synergistic activity observed . In vivo, the combination proved more effective than either drug alone against experimental infections with H . capsulatum, Blastomyces dermatitidis, and Aspergillus species. J Cell Sci, 1983 Jul, 62, 187 - 207 Theoretical analysis of a method for determining the pattern of macromolecular synthesis during the cell cycle; Fraser RS et al.; The dual-labelling centrifugal-elutriation method has been extensively used to study patterns of macromolecular synthesis and accumulation in the yeast cell cycle . Cells are long-term labelled with a radioactive precursor for about 1.5 cycles (a measure of macromolecular mass), then pulse-labelled with a precursor containing a different radioactive isotope for the final 0.1 cycle (a measure of rate of synthesis) . Harvested cells are fractionated into cell cycle stages by centrifugal elutriation, and changes in pulse : long-term labelling during the cycle determined . This pattern of change is compared with theoretical changes in rate : mass calculated for various patterns of synthesis . Using this method, it has been suggested that rates of synthesis and accumulation of several types of RNA and protein all increase exponentially through the cycle . In contrast, experiments using synchronous cultures or zonal centrifugation for cell cycle analysis have suggested other synthetic patterns, including periodic doubling in rate in each cycle . In this paper we analyse whether the dual-labelling, centrifugal-elutriation method is capable of discriminating between exponential and periodic rate-doubling patterns . Three possible sources of imprecision in the method and its application are examined . (1) Theoretical rate : mass curves have been simulated for macromolecules with a wider range of properties than previously considered . It is shown that for some important classes, such as messenger RNAs, with turnover rates in the range measured experimentally, and proteins, differences between rate : mass curves for exponential and periodic rate-doubling models are considerably smaller than previously suggested . (2) Long-term labelling is shown to be an accurate measure of macromolecular mass, but pulse-labelling can be inadequate as a measure of rate of synthesis . The error is greater with RNAs with faster turnover, and again reduces the ability of the method to discriminate exponential and periodic rate-doubling models of synthesis . The error is greater with RNAs with faster turnover, and again reduces the ability of the method to discriminate exponential and periodic rate-doubling models of synthesis . (3) Imperfections in cell cycle fractionation by centrifugal elutriation are examined, and by computer simulation it is shown that these also reduce the ability of the method to distinguish between the two models of synthesis . The three sources of imprecision are cumulative . It is concluded from simulation analyses that the differences between exponential and periodic rate-doubling patterns analysed by the method would be so small as to be almost impossible to establish against the background of error in experimental measurement . We therefore suggest that in practice, the dual-labelling centrifugal-elutriation method is unable to discriminate between the exponentially increasing and periodic rate-doubling models . The mathematical treatment developed in this paper should be applicable to analysis of other methods and cell cycle events. Radiobiologiia, 1983 Jul-Aug, 23(4), 458 - 61 {Modeling the effects of radiation damage to DNA and the genetic risk of radionuclide decay . Dehydrogenation of pyrimidine nucleotides during decay of incorporated 3H}; Korolev VG et al.; Dehydrogenation of DNA pyrimidine nucleotides in thymine positions 5 and 6 and cytosine position 5 is not a drastic lethal damage . Moreover, dehydrogenation of DNA in thymine positions 5 and 6 is not an effective mutagenic lesion . DNA dehydrogenation in cytosine position 5 has proved to be a pronounced mutagenic damage . As to induction of point mutations, 3H is not more harmful than external gamma-radiation given in equivalent doses. Mol Cell Biol, 1983 Jul, 3(7), 1255 - 65 Test for temporal or spatial restrictions in gene product function during the cell division cycle; Dutcher SK et al.; The ability of a functional gene to complement a nonfunctional gene may depend upon the intracellular relationship of the two genes . If so, the function of the gene product in question must be limited in time or in space . CDC (cell division cycle) gene products of Saccharomyces cerevisiae control discrete steps in cell division; therefore, they constitute reasonable candidates for genes that function with temporal or spatial restrictions . In an attempt to reveal such restrictions, we compared the ability of a CDC gene to complement a temperature-sensitive cdc gene in diploids where the genes are located within the same nucleus to complementation in heterokaryons where the genes are located in different nuclei . In CDC X cdc matings, complementation was monitored in rare heterokaryons by assaying the production of cdc haploid progeny (cytoductants) at the restrictive temperature . The production of cdc cytoductants indicates that the cdc nucleus was able to complete cell division at the restrictive temperature and implies that the CDC gene product was provided by the other nucleus or by cytoplasm in the heterokaryon . Cytoductants from cdc28 or cdc37 crosses were not efficiently produced, suggesting that these two genes are restricted spatially or temporally in their function . We found that of the cdc mutants tested 33 were complemented; cdc cytoductants were recovered at least as frequently as CDC cytoductants . A particularly interesting example was provided by the CDC4 gene . Mutations in CDC4 were found previously to produce a defect in both cell division and karyogamy . Surprisingly, the cell division defect of cdc4 nuclei is complemented by CDC4 nuclei in a heterokaryon, whereas the karyogamy defect is not. Arch Biochem Biophys, 1983 Jul 1, 224(1), 342 - 50 Selective loss of mitochondrial genome can be caused by certain unsaturated fatty acids; Graff G et al.; Various unsaturated fatty acids had different effectiveness for maintaining the continued replication of functional mitochondria in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115) . Certain isomers of octadecenoic acid (i.e., cis-9) and eicosatrienoic acid (i.e.,cis-8,11,14) permitted continued replication of mitochondria and provided cultures that contained only 4 to 5% cells that formed petite colonies . On the other hand, cultures grown with cis-12- or cis-13-octadecenoic acid or cis-11,14,17-eicosatrienoic acid, produced a 12- to 16-fold greater frequency of petite mutants (50-60%) after 8 to 10 generations of growth . The production of the petite mutants occurred despite adequate incorporation of these unsaturated fatty acids into cellular phospholipids and an apparently normal ability to undergo the initial steps in the induction of cellular respiration . The evidence suggests that some cellular processes necessary for continued mitochondrial replication depend on the structural features of the fatty acyl chains as well as the overall content of unsaturated fatty acids in membrane phospholipids . Impairment of that process by certain inadequate fatty acids or by an inadequate supply of a suitable fatty acid leads to a permanent loss of the mitochondrial genome from the cells of subsequent generations. J Clin Invest, 1983 Jul, 72(1), 214 - 20 High affinity esterification of eicosanoid precursor fatty acids by platelets; Neufeld EJ et al.; We have examined the relative rates of uptake of several fatty acids into washed, human platelets by measuring incorporation into cellular phospholipids . In the presence of 15 microM fatty acid-free albumin and with radioactive fatty acid concentrations of 5-500 nM, esterification into phospholipid was linear with time and platelet concentration and saturable with respect to fatty acid concentration . Two distinct classes of uptake rate were observed . Arachidonate and 5,8,11,14,17-eicosapentaenoate exhibited high affinity, relatively rapid incorporation into platelet phospholipids at pH 6.5: apparent Michaelis constant (Km) = 30 nM, apparent maximum velocity (Vmax) = 28 pmol/min per 10(9) platelets . Two other eicosanoid precursors, 5,8,11-eicosatrienoate and 8,11,14-eicosatrienoate, exhibited the same Vmax, but Km of 85 and 60 nM, respectively . Under the same conditions, stearate, oleate, and linoleate were incorporated into phospholipids much less efficiently (Vmax approximately 8 pmol/10(9) cells per min, apparent Km greater than or equal to 170 nM) . Qualitatively similar results were found at pH 7.4 . Uptake of radiolabeled, rapid-uptake fatty acids was not diminished by the presence of excess, unlabeled, slow-uptake fatty acids . Thus, the specificity of this esterification system resembles that of the arachidonate-specific, long-chain acyl-CoA synthetase present in platelets . It may represent the expression in vivo of the synthetase, although the apparent affinity of the synthetase for fatty acid is much less . This esterification system probably represents the physiologic mechanism for platelet arachidonate uptake, whereby arachidonate is collected from plasma, despite the fact that its concentration is considerably lower than that of other plasma fatty acids. Arch Biochem Biophys, 1983 Jul 1, 224(1), 1 - 12 Hormonal control of pulmonary surfactant synthesis; Das DK; The specific activities of palmitoyl-CoA synthetase, phospholipase A2, and lysophosphatidylcholine acyltransferase enzymes were low in the lungs of diabetic and hypophysectomized rats as compared to those found in the normal controls . Administration of triiodothyronine (T3), to the diabetic and hypophysectomized rats restored the normal activities of these enzymes . Stimulation of the enzyme activities were also observed when normal rats were injected with the above hormone . The enhancement of the enzyme activities was also found to be dependent on the dose and duration of the hormonal treatment . Optimum levels were achieved at a dose of about 100 micrograms/100 g body weight of T3, 3-4 days after the administration of this hormone . Actinomycin D or cycloheximide abolished the hormone-mediated stimulation of these enzymes in diabetic and hypophysectomized rats . Reduced rate of in vivo palmitoyl-CoA synthetase synthesis was observed in the lungs of diabetic and hypophysectomized animals . Administration of T3 stimulated the rate of synthesis of this enzyme indicating increasing synthesis of this enzyme and not of activation of the pre-existing inactive species . Reduced phospholipid contents, specially decreased amount of lecithin and dipalmitoyl lecithin (DPL) were observed in the lungs of the diabetic and hypophysectomized animals as compared to those in the normal animals . T3 also increased the lecithin and DPL content of the normal rat lungs . These results provide evidence for the involvement of the thyroid hormones in the control of the pulmonary surfactant . The results further suggest that T3 was capable of inducing the enzymes of the "deacylation-reacylation" pathway involved in palmitate incorporation into phosphatidylcholine thereby contributing to the stimulation of dipalmitoyl phosphatidylcholine biosynthesis. Biochem Biophys Res Commun, 1983 Jun 29, 113(3), 941 - 7 Affinity labeling of peptidyl transferase center using the 3' terminal pentanucleotide from amino acyl-tRNA; Perez Gosalbez M et al.; Affinity labeling of proteins in the peptidyl transferase center of eukaryotic ribosomes can be carried out using as a probe p-nitrophenylcarbamyl-amino acyl-tRNA . However, when the reactive p-nitrophenylcarbamyl group is in the amino terminal of the 3' end pentanucleotide derived from amino acyl-tRNA by ribonuclease T1, covalent binding does not take place . An interpretation of the results suggests that the 3' terminal fragment binds to an RNA rich part of the ribosome, which probably forms the P-site in the peptidyl transferase center. Biochim Biophys Acta, 1983 Jun 10, 731(2), 186 - 95 Phosphatidylinositol transfer protein from bovine brain . Substrate specificity and membrane binding properties; Somerharju P et al.; A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H . and Wirtz, K.W.A . (1981) Biochim . Biophys . Acta 649, 521-528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain . The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction . Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred . It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex . In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine . In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein . Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine . This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein . Thus the binding sites for the two phospholipids are mutually exclusive, i.e . phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously . Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system . The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin . In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles . These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes. J Biol Chem, 1983 Jun 10, 258(11), 6979 - 83 The cAMP receptor protein of Trypanosoma cruzi; Rangel-Aldao R et al.; The widest used method to determine cAMP-binding activity in cell-free extracts (Gilman, A . G . (1970) Proc . Natl . Acad . Sci . U.S.A . 67, 305-312) underestimated by about 10-fold the total amount of {3H}cAMP bound by crude extracts of cultured forms of Trypanosoma cruzi (epimastigotes), when compared with the results obtained by the modified Millipore filter technique described by Doskeland and Ueland (Doskeland, S . O., and Ueland, P.M . (1977) Biochem . J . 165, 561-573) . After column chromatography on DEAE-Sephacel, the cAMP-binding activity eluted as a single symmetrical peak at about 210 mM NaCl, totally separated from two peaks of cAMP-independent phosphotransferase activities which eluted, respectively, at 90 and 270 mM NaCl . These two protein kinases showed similar specificities for exogenous substrates, preferring in this order: protamine greater than casein greater than histone H2b . Photoaffinity labeling of cell-free extracts with 8-azido{32P}adenosine 3':5'-monophosphate and autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis specifically labeled only one protein of Mr = 62,000 . From these results it is concluded that T . cruzi epimastigotes possess one single cAMP-binding protein of monomeric Mr = 62,000, not associated with a phosphotransferase activity and probably very different in nature to known regulatory subunits of protein kinases, given the results obtained with the Millipore filtration technique. J Biol Chem, 1983 Jun 10, 258(11), 6750 - 5 Assembly of F1-ATPase in isolated mitochondria; Lewin AS et al.; The assembly of the proton-translocating ATPase complex was studied in isolated mitochondria by incubating yeast mitochondria with radiolabeled precursors of mitochondrial proteins which had been made in a cell-free protein synthesis system . Following such an incubation, the ATPase complex (F1F0) was isolated . Newly assembled F1-ATPase was detected by autoradiography of the isolated enzyme, only peptide subunits which had been made in vitro and imported into the isolated mitochondria could be radioactive . Incorporation of radiolabeled ATPase subunits into the enzyme does not occur in the presence of an uncoupler of oxidative phosphorylation or of a divalent metal chelator, nor does it occur in submitochondrial particles rather than intact mitochondria . Incorporation of labeled ATPase subunits into the enzyme can be completed by unlabeled subunits, provided the unlabeled proteins are added before the mitochondria are incubated with radioactive precursors . These findings suggest that F1-ATPase is assembled from a pool of subunits in mitochondria. Biochemistry, 1983 Jun 7, 22(12), 2945 - 51 Nonstereospecific substrate usage by glyoxalase I; Griffis CE et al.; Glyoxalase I operates on a mixture of rapidly interconverting diasteriomeric thiohemiacetals, formed in a preequilibrium step between glutathione and alpha-ketoaldehyde . That both diasteriomers are directly used as substrates by the enzyme from yeast and from porcine erythrocytes is an outcome of a series of isotope-trapping experiments in which pulse solutions composed of the two diasteriomeric thiohemiacetals, due to {3H}glutathione and phenylglyoxal, are rapidly mixed with chase solutions containing excess unlabeled glutathione and successively increasing concentrations of glyoxalase I . As the enzyme approaches infinite concentration in the chase solution, the radioactivity incorporated into the S-mandeloylglutathione product approaches 100% of the total radioactivity due to both diasteriomers from the pulse solution . The special properties of the active site that allow the enzyme to accommodate both diasteriomeric substrate forms may also account for the fact that the cis and the trans isomers of various para-substituted S-(phenylethenyl)glutathione derivatives are both strong competitive inhibitors of the enzyme . A catalytic mechanism is proposed for glyoxalase I involving catalyzed interconversion of the bound diasteriomeric thiohemiacetals before transformation to final product. Biochim Biophys Acta, 1983 Jun 2, 762(3), 437 - 44 Differentiation under the control of insulin of rat preadipocytes in primary culture . Isolation of homogeneous cellular fractions by gradient centrifugation; Gaben-Cogneville AM et al.; Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats . In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10(-9) M) . During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid: CoA ligase . When VLDL and heparin were added with insulin to the medium, this effect was not potentiated . On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells . With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture . The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions . It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development. Biochem Int, 1983 Jun, 6(6), 743 - 9 Activation of pyridoxamine 5'-phosphate oxidase by aliphatic primary amine; Tsuge H et al.; The reason for observed variable activities of yeast pyridoxaminephosphate oxidase (EC 1.4.3.5; deaminating) was studied . In the presence of an aliphatic primary amine, the pyridoxamine 5'-phosphate oxidase activity was elevated up to 5-fold, whereas pyridoxine 5'-phosphate oxidase was unchanged . Activation resulted from an enhanced Vmax with an almost constant Km . Polyamines (spermine greater than spermidine greater than putrescine) were excellent activators . On the contrary, oxidation of pyridoxine 5'-phosphate or synthetic N-(5'-phospho-4-pyridoxyl)-amino acid was not activated so much . However, a decrease in Km was observed with the increase in the putrescine concentrations. J Lipid Res, 1983 Jun, 24(6), 775 - 80 Lipoprotein lipase: size of the functional unit determined by radiation inactivation; Garfinkel AS et al.; Radiation inactivation was used to determine the functional molecular weight of lipoprotein lipase (LPL) in rat heart and adipose tissues . This technique reveals the size of the smallest unit required to carry out the enzyme function . Supernatant fractions of the tissue homogenates were exposed to high energy electrons at -135 degrees C . LPL activity showed a simple exponential decay in all samples tested . Because changes in nutritional state shift the distribution of LPL between the capillary endothelial and parenchymal cells within heart and adipose tissues, fasted and refed rats were used for the radiation studies . The functional molecular weight was calculated to be 127,000 +/- 15,000 (mean +/- SD) daltons for heart and adipose . Thus, the smallest unit required for enzyme function was the same in both of these tissues and did not vary with nutritional state . The data suggest that, compared with LPL monomer sizes reported in the range 55,000 to 72,000, this active unit constitutes a dimer. Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3318 - 21 Vectorial synthesis of a polysaccharide by isolated plasma membranes; Cabib E et al.; To ascertain the directionality of chitin synthesis by yeast plasma membranes, the external surface of Saccharomyces cerevisiae protoplasts was labeled with ferritin--concanavalin A . After protoplast lysis, plasma membranes were isolated and treated with trypsin to activate chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxy-D-glucosyl-transferase, EC 2.4.1.16) . The membranes were then enrobed in agar and allowed to synthesize chitin from UDP-N-acetylglucosamine . After fixation and embedding in Epon, thin sections were stained for chitin with wheat germ agglutinin--colloidal gold complexes . The chitin marker was found near the ferritin-labeled external face of the membrane--i.e., the polysaccharide was located on the outside of the membrane, as it is in the intact cell . Chitin synthase activity was not detected in intact protoplasts before or after treatment with trypsin . The enzyme became available to trypsin activation after lysis of the protoplasts . Together with similar, previously reported experiments on the inactivation of chitin synthase by glutaraldehyde, these results indicate that the enzyme faces the interior of the cell . We conclude that, both in vivo and in vitro, the synthase receives N-acetylglucosamine residues from UDP-N-acetylglucosamine at the cytoplasmic face of the membrane and transfers them vectorially to a growing chain of chitin that is concomitantly extruded to the outside. J Bacteriol, 1983 Jun, 154(3), 1046 - 53 Control of the ornithine cycle in Neurospora crassa by the mitochondrial membrane; Davis RH et al.; In Neurospora crassa, the mitochondrial membrane separates ornithine used in arginine biosynthesis from ornithine used in the arginine degradative pathway in the cytosol . Ornithine easily exchanges across the mitochondrial membrane under conditions appropriate for synthesis of the immediate biosynthetic product, citrulline . Neither of the two mitochondrial enzymes required for the ornithine-to-citrulline conversion is feedback inhibitable in vitro . Nevertheless, when arginine is added to cells and cytosolic ornithine increases as arginine degradation begins, the rate of citrulline synthesis drops immediately to about 20% of normal (B . J . Bowman and R . H . Davis, Bacteriol . 130:285-291, 1977) . We have studied this phenomenon in citrulline-accumulating strains carrying the arg-1 mutation . Citrulline accumulation is blocked when arginine is added to an arg-1 strain but not to an arg-1 strain carrying a mutation conferring insensitivity of intramitochondrial ornithine synthesis to arginine . Thus, ornithine is evidently unable to enter mitochondria in normal (feedback-sensitive) cells . Other experiments show that cytosolic ornithine enters mitochondria readily except when arginine or other basic amino acids are present at high levels in the cells . We conclude that in N . crassa, the mitochondrial membrane has evolved as a secondary site of feedback inhibition in arginine synthesis and that this prevents a wasteful cycling of catabolic ornithine back through the anabolic pathway . This is compared to the quite different mechanism by which the yeast Saccharomyces cerevisiae prevents a futile ornithine cycle. Nucleic Acids Res, 1983 May 25, 11(10), 3375 - 91 Structure of mouse rRNA precursors . Complete sequence and potential folding of the spacer regions between 18S and 28S rRNA; Michot B et al.; We have determined the complete nucleotide sequence of the regions of mouse ribosomal RNA transcription unit which separate mature rRNA genes . These internal transcribed spacers (ITS) are excised from rRNA precursor during ribosome biosynthesis . ITS 1, between 18S and 5.8S rRNA genes, is 999 nucleotides long . ITS 2, between 5.8S and 28S rRNA genes, is 1089 nucleotides long . Both spacers are very rich in G + C, 70 and 74% respectively . Mouse sequences have been compared with the other available eukaryotes: while no homology is apparent with yeast or xenopus, mouse and rat ITS sequences have been largely conserved, with homologous segments interspersed with highly divergent tracts . Homology with rat is much more extensive for ITS 1 than for ITS 2 . Tentative secondary structure models are proposed for the folding of these regions within rRNA precursor; they are closely related in mouse and rat. J Biol Chem, 1983 May 25, 258(10), 6344 - 51 Characterization of the Rous sarcoma virus transforming gene product; Graziani Y et al.; This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors . The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively) . The radiolabeling of pp60src by {gamma-32P} ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively . Other nucleotides served as weaker inhibitors under the same conditions . The radiolabeling of pp60src by {gamma-32P}GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect . The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants . Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells . In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule . In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors . Similar observations were found with alpha-casein as substrate. Eur J Biochem, 1983 May 16, 132(3), 537 - 44 Interaction of tRNAPhe and tRNAVal with aminoacyl-tRNA synthetases . A chemical modification study; Vlassov VV et al.; The alkylation by ethylnitrosourea of phosphodiester bonds in tRNAPhe from yeast and in tRNAVal from yeast and from rabbit liver and that by 4-(N-2-chloroethyl-N-methylamino)-benzylamine of N-7 atoms of guanosine residues in yeast tRNAVal have been used to study the interaction of these tRNAs with aminoacyl-tRNA synthetases . The modifications occurring at low yield were carried out on 3' and/or 5' end-labelled tRNAs either free or in the presence of cognate or non-cognate synthetases . After splitting of the tRNAs at the alkylated positions, the position of the modification sites in the tRNA sequences were detected by acrylamide gel electrophoresis . It was found that the synthetases protect against alkylation certain phosphate or guanosine residues in their cognate tRNAs . Non-cognate synthetases failed to protect efficiently specific positions in tRNA against modification . In yeast tRNAPhe the cognate phenylalanyl-tRNA synthetase protects certain phosphates located in all four stems and in the anticodon and extra-loop of the tRNA . Particularly strong protections occur on phosphate 34 in the anticodon loop and on phosphates 23, 27, 28, 41 and 46 in the D and anticodon stems . In yeast tRNAVal complexed with yeast valyl-tRNA synthetase the protected phosphates are essentially located in the corner between the amino-acid-accepting and D stems, in the D loop, anticodon stem and in the variable region of the tRNA . Three guanosine residues, located in the D stem, and another one in the 3' part of the anticodon stem were also found protected by the synthetase . In mammalian tRNAVal, complexed with the cognate but heterologous yeast valyl-tRNA synthetase, the protected phosphates lie in the anticodon stem, in the extra-loop and in the T psi arm . The location of the protected residues in the structure of three tRNAs suggests some common features in the binding of tRNAs to aminoacyl-tRNA synthetases . These results will be discussed in the light of informations on interaction sites obtained by nuclease digestion and ultraviolet cross-linking methods. J Biol Chem, 1983 May 10, 258(9), 5424 - 7 Complex formation and intermolecular electron transfer between flavocytochrome b2 in the crystal and cytochrome c; Tegoni M et al.; The present study addresses the question whether tetrameric flavocytochrome b2 in the crystal is catalytically competent and, if so, whether it is possible to prepare a functional complex of the crystalline enzyme with its physiological electron acceptor cytochrome c . By single crystal microspectrophotometry we show that the native reduced enzyme can be oxidized by oxygen or ferricyanide and that the oxidized enzyme can be reduced by the electron donor L-lactate . Reduced cytochrome c appears to diffuse through the liquid channels of flavocytochrome b2 crystals and, at low ionic strength, to accumulate in amounts stoichiometrically equivalent to the enzyme protomers . Both cytochromes can be oxidized by ferricyanide . In the presence of L-lactate, both cytochromes become reduced . Since reduction of cytochrome c by L-lactate requires the catalytic action of flavocytochrome b2, it is concluded that the structure of the crystalline enzyme not only allows for electron transfer from L-lactate to flavin and intramolecular electron transfer from flavin to heme, but also for the formation of a productive complex with cytochrome c. Eur J Biochem, 1983 May 2, 132(2), 249 - 54 Isolation and partial characterization of three histone-specific acetyltransferases from Artemia; Estepa I et al.; Three histone-specific acetyltransferases have been characterized in Artemia by the criteria of cell compartmentation, chromatographic behaviour, substrate specificity and regulatory properties . Acetyltransferase I is a chromatin-bound enzyme with affinity for DNA-cellulose . This enzyme can acetylate histones H1, H3 and H4, but the acetylation of H1 is markedly inhibited in the presence of H4 . Acetyltransferases II and III are cytoplasmic and were resolved by phosphate elution from hydroxyapatite . The isoenzyme II is highly specific for histone H4, whose acetylation is increased in the presence of H1 . The acetyltransferase III is active with the three histone fractions, but its specificity is modulated through the cooperation of H4 (as inhibitor of the acetylation of H1) and H1 (as activator of H4 acetylation) . Spermine was confirmed as a specific activator of the acetyltransferase I, with subsaturating concentrations of H3 as substrate. J Reticuloendothel Soc, 1983 May, 33(5), 401 - 13 Effects of particulate beta-1,3 glucan on human, rat, and guinea pig complement activity; Glovsky MM et al.; Particulate glucan, a beta-1,3-linked polyglucose derived from Saccharomyces cerevisiae, has been demonstrated to have a wide range of immunopotentiating effects . Glucan administration is associated with the modification of a variety of experimentally induced infectious disease states as well as the inhibition of growth of implantable and spontaneous tumors . The present study was designed to evaluate the effect of glucan upon activation of the complement system in rats and guinea pigs . Additional studies were performed to determine the in vitro activating effect of glucan and zymosan on complement activity of human serum . Glucan activated both the classical pathway of normal human sera and the alternate pathways in C2hu-deficient sera in vitro releasing anaphylatoxins such as C3a . The intravenous injection of glucan activated the alternate pathway of guinea pig plasma . The influence of glucan on complement depletion induced by cobra venom factor (CVF) was also ascertained . Complement activation by glucan may contribute, in part, to the enhanced resistance of the host against tumor growth as well as infectious episodes. Cell, 1983 May, 33(1), 25 - 35 The double-strand-break repair model for recombination; Szostak JW et al.; Gene conversion is the nonreciprocal transfer of information from one DNA duplex to another; in meiosis, it is frequently associated with crossing-over . We review the genetic properties of meiotic recombination and previous models of conversion and crossing-over . In these models, recombination is initiated by single-strand nicks, and heteroduplex DNA is generated . Gene conversion is explained by the repair of mismatches present in heteroduplex DNA . We propose a new mechanism for meiotic recombination, in which events are initiated by double-strand breaks that are enlarged to double-strand gaps . Gene conversion can then occur by the repair of a double-strand gap, and postmeiotic segregation can result from heteroduplex DNA formed at the boundaries of the gap-repair region . The repair of double-strand gaps is an efficient process in yeast, and is known to be associated with crossing-over . The genetic implications of the double-strand-break repair model are explored. Neurochem Res, 1983 May, 8(5), 551 - 61 Activation of free fatty acids in subcellular fractions of human skeletal muscle; Trevisan C et al.; In human pathology little is known about the activating enzymes for fatty acids of different carbon chain length . In order to have a better insight into disorders of lipid metabolism in human skeletal muscle, we studied the distribution of acyl-CoA synthetases in muscular subcellular fractions . We find that in muscle mainly long chain fatty acids are activated to CoA esters . Distribution of palmityl-CoA synthetase in subcellular fractions compared with marker enzymes suggested that this enzymatic activity is located only in the outer mitochondrial membrane, in contrast to human liver, where this enzyme is also located in the microsomes . In human skeletal muscle we also found low butyryl-CoA formation, which was limited to the mitochondrial matrix . This site of activation implies that short chain fatty acids may not depend on carnitine for their oxidation in the mitochondrial matrix, in contrast to long chain fatty acids activated in the outer mitochondrial membrane. Anal Biochem, 1983 May, 131(1), 141 - 5 A rapid method for detecting specific RNA transcripts by hybridization to DNA probes in solution; Nobrega FG et al.; A method is described for detecting specific transcripts in crude mixtures of RNA . The method employs hybridization of single-stranded or double-stranded radioactive DNA probes in solution, followed by electrophoretic separation of the hybrid and probe on agarose and visualization by radioautography . The procedure offers the advantages of decreased preparation time and increased sensitivity over currently used methods. J Biol Chem, 1983 Apr 25, 258(8), 5256 - 9 Tertiary structure of the eukaryotic ribosomal 5 S RNA . Accessibility of phosphodiester bonds to ethylnitrosourea modification; McDougall J et al.; The tertiary structure of the eukaryotic ribosomal 5 S RNA was examined using ethylnitrosourea reactivity as a probe for phosphodiester bonds . In three different 5 S RNAs of diverse origin the reactivity was restricted to the same three regions of the sequence, corresponding to residues G 99-A 101, A 88-G 89, and G 75 in the rat 5 S RNA molecule . All of these restricted residues are in highly conserved sequences; five of the residues (G 75, G 89, G 99, A 100, and A 101 in the rat) are present in all 5 S RNAs . The results indicate that, as has been suggested for the secondary structure, the tertiary structure of the 5 S RNA is also highly conserved . Based on these data, a working model for the tertiary structure is suggested which is consistent with previous studies on the structure of the free RNA and 5 S RNA-protein complexes. J Biol Chem, 1983 Apr 25, 258(8), 4937 - 43 Import of proteins into mitochondria . Partial purification of a matrix-located protease involved in cleavage of mitochondrial precursor polypeptides; Bohni PC et al.; Most mitochondrial proteins are synthesized in the cytoplasm as larger precursor polypeptides which are imported into the organelle in an energy-dependent step . The proteolytic conversion of these precursors to their mature size involves a neutral matrix-located protease which has been purified 100-fold from yeast mitochondria . It cleaves the precursors to several imported proteins of the matrix, the mitochondrial inner membrane and the intermembrane space, but is inactive against all mature mitochondrial proteins or against all nonmitochondrial proteins tested so far . As shown in the subsequent report (Cerletti, N., Bohni, P . C., and Suda, K . (1983) J . Biol . Chem . 258, 4944-4949), processing of the cytochrome c oxidase subunit V precursor with the partially purified protease yielded the correct mature NH2 terminus . Precursors to cytochrome b2 and cytochrome c1 (which are imported into the intermembrane space and the outer face of the inner membrane, respectively) are cleaved to intermediate forms which can also be detected as transient forms in vivo . The protease activity has a pH optimum of 7.5 and is inhibited by 1,10-phenanthroline, EDTA, or nucleoside triphosphates, but not by serine-protease inhibitors or by small peptide inhibitors . Its activity can be restored after chelation by excess Co2+ or Zn2+ . The enzyme is coded in the nucleus and is, thus, imported into mitochondria. Nature, 1983 Apr 21, 302(5910), 670 - 6 Molecular analysis of a cell lineage; Nasmyth K; Mating type interconversion has a precise lineage which serves to minimize the time taken for yeast cells to achieve the diploid state . The HO gene either encodes or regulates an endonuclease which initiates the interconversion process . Expression of this gene is switched on during the G1 phase of mother cells and not at all during the cell cycle of their daughters . This behaviour can explain what is known about the lineage. Arch Biochem Biophys, 1983 Apr 15, 222(2), 657 - 60 Sustained oscillations in a reconstituted enzyme system containing phosphofructokinase and fructose 1,6-bisphosphatase; Eschrich K et al.; In a reconstituted open and homogeneous enzyme system containing phosphofructokinase, fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase sustained oscillations could experimentally be generated . The approach is based on a stirred flow-through reaction chamber . The periodic motions of the reactants are mainly caused by the antagonistic allosteric effects of the adenine nucleotides on the activities of the phosphofructokinase and fructose 1,6-bisphosphatase. Biochem Biophys Res Commun, 1983 Apr 15, 112(1), 96 - 101 Role of AMP deaminase reaction in the response of phosphofructokinase to the adenylate energy charge; Yoshino M et al.; The role of NH+4 ion and AMP deaminase reaction in the activation of phosphofructokinase with respect to its response to the adenylate energy charge was investigated using permeabilized yeast cells . (a) Phosphofructokinase and AMP deaminase were activated by the decrease in the adenylate energy charge . The addition of NH+4 further stimulated the phosphofructokinase activity in the presence of intracellular level of K+, and the optimal energy charge value giving the maximal response of the enzyme was shifted from 0.3 to the value above 0.5 . (b) The increase in NH+4 ion produced through the activation of AMP deaminase by spermine which shows no direct action on the phosphofructokinase activity can activate phosphofructokinase with shift of the optimal energy charge value of the enzyme to 0.5 in the presence of K+, whereas the optimal energy charge value for AMP deaminase reaction was not affected by the addition of spermine . Phosphofructokinase can be activated most effectively by the physiological decrease in the energy charge under the condition of increased NH+4 in the presence of K+ . The possibility that the interaction of phosphofructokinase with AMP deaminase under hypoxic condition might be a contributing factor to the Pasteur effect is discussed. J Biol Chem, 1983 Apr 10, 258(7), 4279 - 84 Transfer RNA pyrophosphorolysis with CTP(ATP):tRNA nucleotidyltransferase . A direct route to tRNAs modified at the 3' terminus; Francis TA et al.; The pyrophosphorolysis of tRNA by yeast CTP-(ATP):tRNA nucleotidyltransferase has been studied in an effort to define the behavior of the enzyme and the experimental parameters that lead to net loss of the 3'-terminal nucleotide or to nucleotide exchange . It was found that removal of AMP from the terminus of tRNA proceeded optimally at 1.0 mM PPi; incorporation of 2'- or 3'-dAMP was also studied and shown to proceed optimally at a 6.0 mM concentration of deoxynucleoside triphosphate . CTP was shown to inhibit the pyrophosphorolysis and nucleotide exchange observed when starting from intact tRNA, but apparently not by inhibiting removal of CMP from tRNA missing the 3'-terminal adenosine moiety . The optimized conditions for nucleotide exchange were used for the preparative conversion of tRNAs to species terminating in 2'- and 3'-deoxyadenosine. Anal Biochem, 1983 Apr 1, 130(1), 128 - 33 A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase; Matsubara C et al.; A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide . The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added . The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase . The latter enzyme produces H2O2 . As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid . It was possible to determine free fatty acid in 50 microliters of serum at concentrations ranging from 0.02 to 1.5 mM . The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mM . In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy. Environ Res, 1983 Apr, 30(2), 389 - 92 A comparative study of the effect of triazine herbicides on alcohol dehydrogenases isolated from various sources; Leblova S et al.; The studied herbicides (terbutylazine, simazine) inhibit the activity of plant, animal, and yeast alcohol dehydrogenases . The inhibition constant Ki for alcohol dehydrogenase (ADH) isolated from peas and bakers' yeast equals approximately 10(-4) M, and that for ADH isolated from horse liver is of the order of 10(-5) M . The character of inhibition for all the herbicides studied for the reaction catalyzed by pea, liver, and yeast ADH is always noncompetitive toward ethanol and competitive with respect to NAD . The inhibition constants for the enzyme isolated from peas are pH independent . The interaction constants found for terbutylazine and simazine and for o-phenanthroline, nicotinamide, and ATP indicate that the herbicides are bonded through the metal component of the enzyme, similar to the nicotinamide part of NAD . The interaction constant less than unity found for the herbicide-ATP system indicates that the bonding site in the active center of the enzyme is different for the herbicides and the adenine part of NAD. Mutat Res, 1983 Apr, 114(3), 217 - 67 Mutagenicity and genotoxicity of nitroarenes . All nitro-containing chemicals were not created equal; Rosenkranz HS et al.; The nitrated polycyclic aromatic hydrocarbons constitute a group of chemicals of environmental concern which display a broad spectrum of mutagenic, genotoxic and carcinogenic properties . Some members of the group are the most potent direct-acting bacterial mutagens while others exhibit low levels of potencies which require metabolic activation mixtures . Bacterial mutagenicity is dependent upon reduction of the nitro function . In mammalian cell systems the genetic and genotoxic effects of these nitrated chemicals include the induction of unscheduled DNA synthesis, sister-chromatid exchanges, chromosomal aberrations, gene mutations and cell transformation . The qualitative as well as quantitative expression of these effects is dependent upon the species and tissue of origin as well as culture history of the cell which in turn determine their enzymic capabilities and the conversion of these nitroarenes to ultimate mutagens and genotoxicants . In eukaryotic cells the following bioactivation pathways have been recognized: (a) reduction of the nitro moiety, (b) ring oxidation (the nature of which is influenced by the nitro function) followed by reduction of the nitro group, and (c) ring oxidation without concomitant reduction of the nitro moiety. Anal Biochem, 1983 Apr 1, 130(1), 114 - 9 A microcolorimetric assay of inorganic pyrophosphatase; Shatton JB et al.; A procedure is described for the assay of inorganic pyrophosphatase in tissues by a microcolorimetric procedure, taking advantage of the marked color intensification of phosphomolybdate by malachite green . Conditions are described for optimum enzyme activity, color stability, and sensitivity . With 1-cm cuvettes the AM660 is 100,000, allowing accurate measurement of Pi in the 1-nmol range . Reaction is conducted at 25 degrees C for 10 min in 0.5 ml of a 50 mM histidine buffer, pH 7.2, containing 0.2 mM inorganic pyrophosphate and 4 mM Mg2+, terminated by addition of 0.05 ml 2.4 M HClO4, cooled in ice, and 0.45 ml of color reagent is added . After standing 10 min at 0 degrees C, the contents are transferred to 1-cm cuvettes and the absorbance is read at 660 nm . Blanks are low, nonenzymatic hydrolysis of PPi is negligible, and color is stable without addition of detergents . The high sensitivity makes this procedure well-adapted to measurement of optimal activities in crude tissue preparations. J Biol Chem, 1983 Mar 25, 258(6), 3427 - 30 Import of proteins into mitochondria . In vitro studies on the biogenesis of the outer membrane; Gasser SM et al.; The yeast mitochondrial outer membrane contains a major 29-kilodalton protein which is encoded in the nucleus . When this polypeptide is synthesized in vitro and then incubated with isolated yeast mitochondria, it binds to the organelles and becomes resistant to externally added trypsin . Post-translational insertion of the 29-kilodalton protein in vitro is not accompanied by NH2-terminal processing, appears to require neither ATP nor a transmembrane electrochemical potential, and is also observed with purified outer membranes, but not with yeast microsomes . In vitro insertion of the 29-kilodalton polypeptide is thus in several respects different from that of polypeptides destined for the internal compartments of the mitochondrion. J Mol Biol, 1983 Mar 25, 165(1), 59 - 77 Topological modifications and template activation are induced in chimaeric plasmids by inserted sequences; Carnevali F et al.; The effect of the insertion of foreign genes or gene systems in closed DNA domains has been investigated in vitro in purified systems . We observe that in chimaeric plasmids two apparently independent classes of modifications, (1) functional and (2) topological, do take place in defined instances . (1) Among the screened yeast gene systems, examples have been found of DNA sequences that upon insertion cause activation of in vitro transcription of distant genes . (2) Foreign DNA sequences may lead to new topological features of the harbouring plasmids; it is shown that more than one S1-sensitive secondary structure may be contemporaneously present on the same chimaeric plasmid . DNA superhelicity is a prerequisite of these modifications . The two classes of effects (1) functional and (2) topological are not a priori directly related one to the other but appear to be two independent consequences of the same cause: the insertion of foreign DNA sequences into closed DNA domains . These observations suggest a regulatory model of gene expression based on alternative topologies of closed DNA domains. Nucleic Acids Res, 1983 Mar 25, 11(6), 1725 - 34 Nucleotide sequence of the transcriptional initiation region of Dictyostelium discoideum rRNA gene and comparison of the initiation regions of three lower eukaryotes' genes; Hoshikawa Y et al.; The 5' end of the rRNA precursor of D . discoideum was mapped on a cloned rDNA by S1 nuclease protection mapping, and the sequence of about 1240 nucleotides surrounding the transcriptional initiation site of the rRNA gene has been determined . Repeated sequences consisting of 16 nucleotides appeared in the region upstream from the initiation point . Comparison of the nucleotide sequences around the initiation site of rRNA genes in three lower eukaryotes, D . discoideum, Saccharomyces cerevisiae and Tetrahymena pyriformis, indicated that there was little similarity in the nontranscribed spacer regions, but in the transcribed spacer regions near the initiation point, very similar sequences consisting of 9 nucleotides were found. J Biol Chem, 1983 Mar 25, 258(6), 3655 - 60 Manipulation of the observed kinetic phases in the refolding of denatured ferricytochromes c; Brems DN et al.; The refolding of guanidine hydrochloride-denatured horse heart ferricytochrome c at pH 7.0 and 23 degrees C occurs in three kinetic phases as observed by stopped flow measurements using changes in Soret absorbance or in tryptophan fluorescence . The three kinetic phases have time constants of 10 +/- 5 ms, 240 +/- 30 ms, and 13 +/- 3 s accounting for 15 +/- 5%, 70 +/- 5%, and 15 +/- 5% of the total reaction, respectively . The intermediate kinetic phase can be selectively eliminated by conducting the refolding measurements at pH 5.0 . Both the intermediate and slow kinetic phases can be eliminated by conducting the refolding measurements either at pH 7.0 or at pH 5.0 in the presence of an excess of an extrinsic ligand for an axial position of the heme iron . Similar results are obtained using tuna heart ferricytochrome c except that the fractional reaction in the fast and intermediate kinetic phases at pH 7.0 in the absence of extrinsic ligand are 29 +/- 2% and 58 +/- 2%, respectively . We suggest that both the intermediate and slow kinetic phases are generated by proline peptide isomerization occurring during and prior to the refolding procedure, respectively, and that their occurrence is dependent upon the conformation of the denatured protein and upon the ligation of methionine 80 in the folded product, respectively. Biochim Biophys Acta, 1983 Mar 16, 743(2), 246 - 55 Assignment of hyperfine shifted resonances in high-spin forms of cytochrome c peroxidase by reconstitutions with deuterated hemins; Satterlee JD et al.; Assignments of hyperfine shifted proton resonances for the high-spin forms of cytochrome c peroxidase (EC 1.11.1.5) have been made (cytochrome c peroxidase, cytochrome c peroxidase-F) employing the technique of reconstituting the apoprotein with specifically deuterated protohemin IX derivatives . The results show that the heme methyl group pattern differs significantly from similar assignments made for metmyoglobin . In cytochrome c peroxidase the methyl pattern is 5 greater than 1 greater than 8 greater than 3 . For cytochrome c peroxidase-F the pattern is 5 greater than 8 greater than 1 greater than 3, but the resonances are not shifted as far downfield and they exhibit a narrower spread . For myoglobin the relative methyl ordering has previously been shown to be 8 greater than 5 greater than 3 greater than 1 . Several conclusions have been reached, including confirmation of the essential correspondence between the solution- and crystal-derived data for several heme crevice structural features . The pH dependence of the cytochrome c peroxidase-F methyl resonances is also presented and is shown to differ from native peroxidase . For cytochrome c peroxidase-F smooth, continuous titrations are observed with no evidence of the second conformation which was found for the native enzyme. Mutat Res, 1983 Mar, 116(3-4), 323 - 31 Genetic and biochemical studies on perchloroethylene 'in vitro' and 'in vivo'; Bronzetti G et al.; Perchloroethylene (PCE) was tested in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system (S9) and 'in vivo' by the intrasanguineous host-mediated assay . In addition, enzyme alteration studies were performed in mice non-pretreated or pretreated with phenobarbital + beta-naphthoflavone . PCE did not induce any genetic effect either 'in vitro' or 'in vivo' . In the suspension test, PCE was more toxic without metabolic activation and less toxic with mammalian microsomal activation . The enzymatic determinations showed an increase of the aminopyrine demethylase activity and of the level of cytochrome P-450. Biochim Biophys Acta, 1983 Feb 28, 743(1), 121 - 8 Sepharose-stearate as substrate for rat liver long-chain fatty acyl-CoA synthetase; Rosen G et al.; Stearic acid coupled covalently to Sepharose 6B serves as substrate for thioesterification catalyzed by rat liver long-chain fatty acyl-CoA synthetase (ATP-forming) (EC 6.2.1.3) . Availability as substrate is dependent upon the conservation of the free omega-terminal in addition to that of the free carboxyl function . The enzymatic overall formation of matrix-acyl-CoA in the presence of ATP and CoA as cosubstrates conforms to the stoichiometry reported for thioesterification of the free long-chain fatty acyl substrate . The preformed matrix-acyl-CoA serves as substrate for the backward synthetase reaction in the presence of AMP and PPi . The apparent Km values for ATP and CoA in the presence of the acyl matrix are similar to the respective Km values observed in the presence of the free acid substrate . The apparent Km for the acyl matrix is 10-fold higher (0.5 mM) than the apparent Km value for the free acid . The feasibility of enzymatic thioesterification of bound long-chain fatty acids implies that the exact nature of the bulky chain situated between the carboxy and omega-terminal plays a secondary role in defining the fatty acyl substrate specificity for long-chain fatty acyl-CoA synthetase . Also, dissociation of bound long-chain fatty acids does not constitute an obligatory preliminary step to fatty acid thioesterification. J Biol Chem, 1983 Feb 10, 258(3), 1991 - 9 Functional domains in introns . RNA processing intermediates in cis- and trans-acting mutants in the penultimate intron of the mitochondrial gene for cytochrome b; Lamb MR et al.; The penultimate intron of the split mitochondrial gene (cob) for apocytochrome b of Saccharomyces cerevisiae is of particular interest; it contains a long unassigned reading frame, is present in both long form (six exons) and short form (three exons) of the gene, and a product expressed from it is required for the removal of its transcript and that of an intron in the transcript of the oxi3 gene . Complementation analysis shows mutants in this intron to be either cis-dominant or transrecessive . Cis-dominant mutants are located in the first third (approximately 350 base pairs) of the open and near the 3'-end of the closed reading frame, while trans-recessive mutants are scattered throughout the remaining two-thirds (approximately 750 base pairs) of the open frame . Mutants in both classes exhibit the same pattern of splicing defects in their transcripts, but for different reasons . Those in the trans-recessive class lack a functional maturase (probably a protein of Mr = 27,000) encoded wholly within the 3'-terminal segment of the intron, and for this reason also fail to express oxi3 . In contrast, cis-dominant mutants are incapable of providing the splicing complex with a substrate of appropriate 2 degrees structure . They also accumulate a novel transcript, 1900 nucleotides long, which contains the intron fused to the downstream (3') exons . This may reflect an inability of the splicing complex to complete the normal sequence of cleavage of the intron at its downstream junction and the ligation of the two exonic moieties. Eur J Biochem, 1983 Feb 1, 130(2), 261 - 8 A study of some molecular and kinetic properties of two tRNA methyltransferases from mouse plasmocytoma; Nau F et al.; A tRNA(adenine-1)methyltransferase and a tRNA(cytosine-5)methyltransferase have been partially purified from mouse plasmocytoma MOPC 173 . Their apparent Mr are 200000-230000 and 110000-140000, respectively, as determined by gel filtration and density gradient centrifugation . Both enzymes exhibit maximum activity in the presence of high concentrations of monovalent cations (0.175 M and 0.25 M KCl, respectively) and in the absence of magnesium . Their kinetic constants have been determined at various KCl concentrations, with several tRNA species as substrates . These constants may differ by more than one order of magnitude, depending upon the substrate used, and they are strongly dependent upon the ionic concentration as well . The possibility that the tRNA(adenine-1)methyltransferase from mouse plasmocytoma is different from the homologous enzyme purified from a normal rat tissue {Glick, J . M . and Leboy, P . S . (1977) J . Biol . Chem . 252, 4790-4795} is discussed. Cryobiology, 1983 Feb, 20(1), 61 - 77 Osmotic response of individual cells during freezing . I . Experimental volume measurements; Schwartz GJ et al.; The osmotic response of yeast to freezing was measured as a function of cooling rate and degree of extracellular supercooling . Thirteen experimental trials were conducted on a cryomicroscope in which incremental size changes of individual cells were recorded photographically, and the corresponding volume variations were measured using a digital computer image analysis algorithm . Plots were obtained of normalized cell volume as a function of temperature . Cellular dehydration during freezing was progressively inhibited with increasing values of cooling rate and extracellular supercooling . Normalized cell volume changes were not a function of the relative initial cell size . A constant volume plateau occurred for conditions under which intracellular ice formation was expected. Proc Natl Acad Sci U S A, 1983 Feb, 80(4), 1096 - 100 Detection of autonomous replicating sequences (ars) in the genome of Epstein-Barr virus; Henry BE 2nd et al.; Epstein-Barr virus (EBV) DNA was analyzed for the presence of autonomous replicating sequences (designated ars) in a eukaryotic system consisting of a uracil auxotroph of Saccharomyces cerevisiae, YNN27, and a pBR322 hybrid plasmid, YIp5, containing the yeast uracil gene but apparently lacking a eukaryotic origin of replication . Cloned EBV DNA EcoRI restriction fragments, A, B, and DIJhet, were judged to function in this capacity by their ability to convert YNN27 cells to the uracil phenotype after transformation with each EBV-specific fragment ligated into YIp5 . Additional analyses to confirm and to specify further the location of the ars were performed by cleavage of EcoRI fragments A and B into smaller BamHI fragments, which were subsequently cloned in YIp5 and tested for their ability to function as ars . BamHI fragment X, obtained from EcoRI fragment A, and BamHI fragment R, obtained from EcoRI fragment B, showed ars behavior . The successful recovery of the appropriate virus DNA segments in plasmid form from transformed yeast cells and the ability of these yeast cells to be propagated further substantiated the ars capability of the three EBV fragments. Ann Biomed Eng, 1983, 11(5), 361 - 84 Computer simulation of metabolism in palmitate-perfused rat heart . I . Palmitate oxidation; Kohn MC et al.; A computer model of the fatty acid oxidation pathway in perfused rat heart was constructed . It includes uptake, activation, and beta-oxidation of fatty acids, triglyceride synthesis and hydrolysis, and carnitine-dependent transport of acyl groups across the mitochondrial membrane under pseudosteady state conditions . Fatty acid utilization may be limited by beta-oxidation in hypoxia or ischemia but probably not in aerobic conditions . Nonesterified fatty acids bound to proteins are found to be metabolically available . The model predicts that stearate, but not palmitate, can support the highest observed respiration rate for perfused rat heart without supplementation by other substrates . Fatty acids are preferentially oxidized rather than being stored as triglycerides because the cystosolic acyl CoA level is lower than the Km for triglyceride synthesis . It is suggested that feedback inhibition of triglyceride lipase regulates utilization of triglycerides as fuel in aerobic hearts. Placenta, 1983, 4 Spec No, 499 - 513 Characterization of three aminopeptidases purified from human placenta; Lampelo S et al.; Three aminopeptidases purified from the human placenta were characterized and compared with each other . Aminopeptidase II1 preferred L-arginine- and L-lysine-beta-naphthylamides or p-nitroanilides as substrate, with low or negligible hydrolysis of other amino acid derivatives . It was inhibited by L-arginine, L-lysine and L-methionine . This enzyme activity was highly sensitive to heat treatment, N-ethylmaleimide, p-chloromercuribenzoate, puromycin, bestatin, epsilon-amino-n-caproic acid (EACA) and EDTA . After EDTA, this enzyme could be reactivated by Co2+ . It is concluded that aminopeptidase II1 is identical with arginine aminopeptidase (EC 3.4.11.6) from other mammalian tissues . Aminopeptidase II2 preferred L-alanine-beta-naphthylamide and p-nitroanilide as substrates . It was also able to hydrolyse L-leucine, L-arginine, L-methionine and L-lysine derivatives but only very weakly L-cystine and Bz-L-cysteine substrates . This enzyme was inhibited by L-arginine, L-alanine, L-lysine and most strongly by L-leucine and L-methionine . It was resistant to bestatin and heat treatment but sensitive to EACA . EDTA caused a marked suppression, which could be prevented by Co2+ and Zn2+ . These characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-) found in other tissues . The third enzyme was the only one clearly particle bound and was therefore called PB-aminopeptidase . It preferred L-leucine derivatives as substrate but also readily hydrolysed other amino acid-beta-naphthylamides and p-nitroanilides including L-cystine and Bz-L-cysteine substrates . Among the amino acids L-cysteine, L-leucine and L-methionine were inhibitory . Bestatin and thiol reagents were without effect and EACA was only moderately inhibitory . EDTA caused a strong suppression, which could be prevented by Co2+ and Zn2+ . These properties are equal to those previously described for the placental cystine aminopeptidase (oxytocinase) (EC 3.4.11.3) . All three enzymes had an optimum close to neutral pH but apparent differences in their Km and Vmax values with various substrates . These findings suggest that the three purified aminopeptidases are distinct enzymes . Two of these (aminopeptidases II1 and II2) have not previously been isolated and characterized in the human placenta. Adv Exp Med Biol, 1983, 163, 139 - 48 Evaluation of folylpolyglutamates by electrophoretic separation of fluorodeoxyuridylate-thymidylate synthase-methylenetetrahydrofolate complexes; Priest DG et al.; The chain length of specific reduced folylpolyglutamates has been estimated by incorporation of the 5,10-methylenetetrahydrofolate form of the cofactor into a stable ternary complex with L . casei thymidylate synthase and tritiated fluorodeoxyuridylate followed by electrophoretic separation based on charge differences in complexed polyglutamates . The method is also applicable to tetrahydrofolate polyglutamates after conversion to the active cofactor form by introduction of formaldehyde . The method can be used to analyze less than one pmole of folylpolyglutamate and can be applied to evaluation of tissue polyglutamates or to monitor relevant enzyme c