Acta Histochem Suppl, 1981, 23, 29 - 36 Interpretation of morphological data obtained by freeze-fracturing at very low temperatures; Sleytr UB et al.; In freeze-fracture replication the greatest variety of methods exists for the fracturing process itself . The present stage of knowledge allows to evaluate fracture faces obtained by freeze-cleaving at temperatures down to 4 K . Under condensation free conditions the possibility for artefact formation will be restricted to the fracturing and replication process . Experiments on model systems and complex biological systems show that many fine structural details seen on freeze-fractured replicas have to be interpreted as the result of a multi-event process involving plastic deformation, elastic recontraction, collapse phenomena and thermal load induced alterations during replica formation . With some structures the degree of deformation may be reduced by chemical fixation procedures. DNA, 1981, 1(1), 1 - 9 Multigene family of actin-related sequences isolated from a soybean genomic library; Nagao RT et al.; We have investigated the actin-related sequences in soybean using heterologous actin DNA probes from Dictyostelium, Drosophila, and yeast . Southern blot analysis of restriction digests of soybean DNA indicates that actin is encoded in a small multigene family . In order to isolate individual members of this gene family, we have constructed a soybean genomic library in the lambda vehicle Charon 4A . A partial characterization of this library shows it to be nearly complete . We have isolated from this library a number of recombinant clones that hybridize to actin-coding sequences from all three heterologous probes . We have identified the fragments containing the actin-related sequences on the physical maps of two of these clones lambda SAc1 and lambda SAc3 . These fragments were subcloned in the plasmid vehicle pBR322 . Using electron microscope heteroduplex mapping we show that the subclones, pSAc1 and pSAc3, share homology with the entire actin-coding sequence (1.1 kb) of Drosophila and Dictyostelium . Furthermore, pSAc1 and pSAc3 have additional homology of approximately 0.22 kb at the 5' ends of their coding sequences . No homology is detected in the 3' flanking regions of these clones . The actin sequence in pSAc1 contains an interruption of approximately 0.30 kb located 0.39 kb from the 5' end of the actin polypeptide coding region. Dev Pharmacol Ther, 1981, 3(1), 55 - 64 Effect of dexamethasone on the synthesis of dipalmitoyl phosphatidylcholine; Das DK et al.; Glucocorticoids are known to enhance surfactant production by stimulating the formation of phosphatidylcholine . This study was performed to determine the effect of steroids on one of the pathways involved in the incorporation of palmitic acid into phosphatidylcholine--the 'deacylation-reacylation pathway' . Palmitoyl CoA synthetase, lysopalmitoyl cholineacyltransferase and phospholipase A2 were enhanced significantly as a result of direct administration of dexamethasone into 27-day fetal rabbits . Cycloheximide inhibited the steroid induced enhancement of activity . Immunologic studies demonstrated an accelerated rate of synthesis of palmitoyl CoA as a result of the dexamethasone treatment . The data suggest that steroids are capable of inducing enzymes of the 'deacylation-reacylation pathway' involved in palmitate incorporation into phosphatidylcholine thereby contributing to the acceleration of dipalmitoyl phosphatidylcholine biosynthesis. Pediatr Pharmacol (New York), 1981, 1(3), 209 - 14 Effect of aminophylline on the synthesis of dipalmitoyl phosphatidyl choline in fetal rabbit lung; Ayromlooi J et al.; Aminophylline (A) administration to pregnant rabbits resulted in accelerated formation of phospholipids, the known important components of pulmonary surfactant {1} . The present study was undertaken to determine the effect of A on one of the pathways involved in the incorporation of palmitic acid into phosphatidyl choline (PC)--the "deacylation-reacylation pathway." A was injected intraperitoneally into rabbit fetuses at 27 days of gestation and its effect on palmitoyl CoA synthetase (PCS) and lysopalmitoyl choline acyl transferase (LPC-AT) were studied . Only LPC-AT was enhanced significantly (P less than 0.05) as a result of direct administration of A . This study supports the suggestions by previous investigators that antenatal administration of aminophylline may prove to be an effective means of enhancing lung maturation by stimulating the formation of pulmonary surfactant production before premature delivery . However, the dose that was used in this experimental study was far more than the usual clinical dose that had been suggested previously. Acta Biol Med Ger, 1981, 40(10-11), 1393 - 6 Initiation of selective proteolysis by metabolic interconversion; Holzer H; After the addition of glucose to acetate- or ethanol-grown yeast cells a small group of selected enzymes is rapidly inactivated . This phenomenon has been called "catabolite inactivation" . Among other enzymes participating in gluconeogenesis, fructose-1,6-bisphosphatase is inactivated during this catabolite inactivation process . It was shown by FUNAYAMA et al . (Eur . J . Biochem . 109, 61-66 (1980)) that the mechanism of inactivation is proteolysis . In the present paper evidence is presented that after addition of glucose a covalent conversion of the enzyme protein by phosphorylation of a serine-residue initiates its subsequent proteolysis . It is suggested that the covalent modification triggered by glucose and/or products of its catabolism renders the enzyme susceptible to proteinases and thereby initiates proteolysis of a selected enzyme without the necessity of a specific proteinase present. Biokhimiia, 1981 Jan, 46(1), 33 - 9 {Comparative properties of metals activating inorganic pyrophosphatase}; Volk SE et al.; Using an ion-selective electrode, the complex formation in a Zn2+-PPi system at pH 6,52 and ionic strength of 0,1 M, t=25 degrees, was studied . The concentrations of the ZnPPi complex (true substrate) and free cation Zn2+ (activator) were calculated from the values of the dissociation constants for the ZnPPi and Zn2PPi complexes . The activating effects of these complexes on inorganic pyrophosphatase were similar to those exerted by Mn2+ and Zn2+ . The kinetic patterns of both complexes revealed three main sites for cation binding to the enzyme -- substrate complex . The excess of substrate and activator inhibited the enzymatic reaction . The Km and V values for ZnPPi are 4.1 and 1.2 times lower than those for MgPPi . Thus, Zn2+ appear to be a more efficient activator than Mg2+ at non-saturating concentrations . The data obtained suggest that the occurrence of the limiting step is practically the same for both metals. Nucleic Acids Res, 1980 Dec 20, 8(24), 5993 - 6005 Nucleotide sequence through the 18S-28S intergene region of a vertebrate ribosomal transcription unit; Hall LM et al.; We have determined the nucleotide sequence of part of a cloned ribosomal transcription unit from Xenopus laevis extending from the 3' region of the 18S gene through the 18S-28S intergene region into the start of the 28S gene . The 18S 3' region possess two tracts of high homology with the corresponding segments of other eukaryotic 18S genes (yeast and Bombyx mori) separated by a tract of low homology which in X . laevis is rich in G plus C . The first internal transcribed spacer, between the 18S and 5.8S genes, is 557 nucleotides long, very rich in G plus C (84%) and shows no sequence homology with the corresponding yeast sequence . The 5.8S rRNA sequence is revised slightly in the light of the DNA sequence . The second internal transcribed spacer, between the 5.8S and 28S genes, is 262 nucleotides long and is even richer in G plus C (88%) than the first internal spacer . 28S rRNA starts with the sequence pUCAG . This is encoded at the first of three closely linked TCAG sites in rDNA. Nucleic Acids Res, 1980 Dec 20, 8(24), 6043 - 58 The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse; Urano Y et al.; Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced . The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA . Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG--- . Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's . A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site . No such characteristic sequences were found downstream this site . Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Biochim Biophys Acta, 1980 Dec 11, 610(2), 425 - 9 Influence of pH and length of post-treatment incubation on bleomycin-induced DNA damage; Moore CW et al.; The dependence of the extent of DNA damage by anticancer bleomycin on pH and length of post-treatment incubation was studied in yeast . Bleomycin was always removed from cells after 20-min exposures, and cells were washed prior to incubation in non-nutrient buffer . Following exposures of late stationary-phase cells to the very low dose of only 3 micrograms/ml, 1.5 h incubation in non-nutrient buffer, pH 5, had hardly any effect on profiles derived from alkaline sucrose gradient sedimentation of nucleic acids released from spheroplasts . In contrast, after incubation of cells for 1.5 h in buffer, pH 7, DNA was all low molecular weight . Thus, even after extensive washing of cells, pH strongly influences the drug's action on DNA . At pH 5, washed cells were increasingly susceptible to DNA damage up to 26 h in non-nutrient buffer. J Biol Chem, 1980 Dec 10, 255(23), 11454 - 63 Comparative studies of histone acetylation in nucleosomes, nuclei, and intact cells . Evidence for special factors which modify acetylase action; Garcea RL et al.; We have studied the pattern of histone acetylation in intact rat hepatoma tissue culture (HTC) cells, in isolated HTC nuclei, and in chromatin prepared from these cells . The results have been compared with the histone acetylation observed in a reconstituted in vitro system consisting of a variety of purified soluble nucleosomal substrates, {3H}acetyl-CoA, and one of two different purified histone N-acetyltransferases . Acetylase A, a highly purified nuclear enzyme, catalyzed the acetylation of 1) nucleosomally bound histones in the order H4 > H2a = H2b > H3, and 2) free histones in the order H4 > H3 > H2b > H2a . Acetylase B, a cytoplasmic enzyme, modified only free histone H4, and it failed to acetylate histones in nucleosomes . The pattern of histone acetylation obtained by in vitro reaction of purified nucleosomes with the purified nuclear acetylase A differed considerably from the corresponding patterns obtained either by acetate labeling of intact cells, or by the acetyl-CoA labeling of nuclei and crude preparations of nucleosomes, as catalyzed by endogenous chromatin-bound acetylase(s) . The most striking difference was in the relative preference for acetylation of histone H4 versus acetylation of histone H3: with the purified acetylase, histone H4 in nucleosomes was acetylated to a much greater extent than was histone H3, whereas the reverse preference was found with the endogenous acetylase(s) . This result suggests that either a second nuclear acetylase enzyme, or a separate cofactor for acetylase A, is required for histone H3 acetylation in vivo . In support of this view, we find that the acetylation of histones H4, H2a, and H2b in nuclei is inhibited by urea, salt, or N-ethylmaleimide treatments to a very different extent than is the acetylation of histone H3 . By comparing n-butyrate-treated HTC cells with untreated cells, classes of nucleosomes specially accessible and inaccessible to acetylation can be distinguished (Cousens, L . S., Gallwitz, D., and Alberts, B . M . (1979) J . Biol . Chem . 254, 1716-1723) . Both types of special nucleosomal reactivities were present in isolated nuclei, but were lost as nucleosomes were purified from these cells . OUr data thus suggest the existence of labile specificity factors or structures, which guide the acetylase(s) to restricted groups of otherwise similar nucleosomes in vivo. J Biol Chem, 1980 Dec 10, 255(23), 11448 - 53 Extensive purification of histone acetylase A, the major histone N-acetyl transferase activity detected in mammalian cell nuclei; Belikoff E et al.; A concentrated, DNA-free extract of histone acetylase A was prepared from calf thymus tissues in two simple steps, which exploit the ability of polyethylene glycol to precipitate both nucleic acids and proteins from solutions containing high concentrations of salt (Alberts, B., and Herrick, G . (1971) Methods Enzymol . 21, 198-217) . This extract was then chromatographed on four successive columns . The use of 75 microgram/ml of insulin as a carrier protein in all of these later steps, plus the inclusion of 1 M urea in some column buffers, has been useful in improving both the yield and reproducibility of the purification . The highly active enzyme obtained has a molecular weight of about 70,000, and the best fractions could be about 30% pure . Our data indicate that the acetylase A is only a very minor protein in cells, being present in perhaps a few thousand molecules per cell. Mutat Res, 1980 Dec, 74(6), 439 - 58 Quantitative measures of mutagenicity and mutability based on mutant yield data; Eckardt F et al.; We described how mutant yield data (mutants per cell treated) can be used both to compare the mutagenicity of different mutagens, and to characterize the mutability of different cell types . Yield curves reveal the net effect of the lethal and genetic actions of mutagens on cells . Normally, yields are the quantities measured in assays for mutagenesis, and rectilinear plots of such data baldly reveal the amount of experimental error and the extent of actual mutant induction above the background level . Plots of yield versus lethal hits can be used to quantify the relative mutagenic efficiency (RME) of agents whose physical exposure doses otherwise would be incommensurable, as well as the relative mutability (Rmt) of different strains to the same mutagen . Plots of yield versus log dose provide an unambiguous way of assessing the relative mutational sensitivities (Rms) and mutational resolutions (Rmr) of different strains against a given mutagen . Such analysis is important for evaluation of the relative merits of excision-proficient and excision-deficient strains of the same organism as mutagen-testing systems . The mathematical approach outlined here is applied, by way of example, to measurements of UV and 4-NQO induced mutagenesis in both repair-deficient and repair-proficient haploid strains of the yeast Saccharomyces cerevisiae. Nature, 1980 Nov 27, 288(5789), 404 - 6 Conservation and rearrangement of mitochondrial structural gene sequences; Macino G et al.; Mitochondria contain the simplest DNA molecules that are present in eukaryotes . Mitochondrial DNA (mtDNA) is easily purified, and is an important model system for studying eukaryote gene structure and basic molecular processes . The protein sequences of mitochondrial gene products have been shown to be conserved from yeast to man, and there are definite similarities at the DNA sequence level . In contrast, the overall organization of the mitochondrial genome is drastically different in these organisms . To understand this, we need to extend work on mtDNA to a wider range of species . We have chosen to study the mtDNA of Aspergillus nidulans because a particularly comprehensive analysis of this system can be achieved using genetics as well as biochemistry, and like most eukaryotes it is an obligate aerobe, whereas Saccharomyces cerevisiae is not . We have investigated whether defined pieces of particular yeast mitochondrial genes show enough homology to Aspergillus mtDNA fragments to enable the corresponding Aspergillus genes to be located on the physical map . The results reported here show that this is the case for all five genes tested, and present the first data on the physical organization of the structural genes in the mitochondrial genome of A . nidulans. J Biol Chem, 1980 Nov 25, 255(22), 10717 - 20 ADP-ribosylation of elongation factor 2 by diphtheria toxin . Isolation and properties of the novel ribosyl-amino acid and its hydrolysis products; Van Ness BG et al.; We have isolated the novel amino acid, amino acid X, in elongation factor 2 (EF-2) which is ADP-ribosylated by diphtheria toxin (Robinson, E . A., Hendriksen, O., and Maxwell, E . S . (1974) J . Biol . Chem . 249, 5088-5093) . The pure ribosyl-amino acid was obtained in milligram quantities following sequential enzymatic degradation of yeast ADP-ribosyl-EF-2 (Van Ness, B . G., Howard, J . B., and Bodley, J . W . (1978) J . Biol . Chem . 253, 8687-8690) with an overall yield of 30 to 60% . In the course of acid hydrolysis, the ribosyl-amino acid was converted first to an intermediate compound which did not contain ribose . Further acid hydrolysis converted the intermediate to amino acid X and ammonia . Alkaline hydrolysis of the ribosyl-amino acid produced a different intermediate which contained ribose . We have isolated the two hydrolysis intermediates and on the basis of the properties of all four compounds we conclude that the ribosyl-amino acid contains an amido function which is not involved in a glycosidic linkage and a net positive charge of 2 at acid pH . We believe that the amino acid amide occurs in EF-2 and for this compound we propose the trivial name diphthamide . Acid hydrolysis of diphthamide yields amino acid X for which we propose the trivial name diphthine. J Biol Chem, 1980 Nov 25, 255(22), 10563 - 5 Mapping of mitochondrial structural genes in Neurospora crassa; Macino G; A hybridization method has been employed to study the organization of the mitochondrial genome of Neurospora crassa . The method involves the use of 5' end-labeled single-stranded restriction fragments obtained from cytoplasmic "petite" strains of Saccharomyces cerevisiae known to contain single mitochondrial genes . The presence and localization of genes homologous to Subunits 1, 2, and 3 of cytochrome oxidase, cytochrome b and Subunit 6 of the ATPase is thus established for the mitochondrial genome of N . crassa. Biochim Biophys Acta, 1980 Nov 18, 602(3), 578 - 90 Topography of phosphatidylcholine, phosphatidylethanolamine and triacylgycerol biosynthetic enzymes in rat liver microsomes; Ballas LM et al.; The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide . Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran . Dextran-maleimide caused 52% inactivation of the sn-glycerol-3-phosphate acyltransferase . Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9) . These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites . Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane . These data indicate that the active sites of thease enzymes are located on the external surface of microsomal vesicles . It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. Nucleic Acids Res, 1980 Nov 11, 8(21), 5095 - 111 The electrostatic molecular potential of tRNAPhe . IV . The potentials and steric accessibilities of sites associated with the bases; Lavery R et al.; The sites of the 76 nucleic acid bases of tRNAPhe potentially reactive towards electrophiles are studied by calculations on the associated molecular electrostatic potentials and the static steric accessibilities . Each of these sites is treated in its environment within the macromolecule . The influence of various schemes of screening by countercations of the backbone phosphates on the electrostatic potentials is investigated . The possible significance of the potentials and accessibilities in connection with observed chemical reactivities is discussed. J Gen Microbiol, 1980 Nov, 121(1), 243 - 7 Genetic evidence for a second asparaginase in Aspergillus nidulans; Arst HN Jr et al.; The apnA1 mutation strongly reduces L-asparagine utilization in Aspergillus nidulans . The ahrA1 mutation, leading to loss of an L-asparaginase (Drainas et al., 1977), eliminates residual L-asparagine utilization in double mutant strains also carrying apnA1 . This additivity suggests that A . nidulans, like Saccharomyces cerevisiae (Jones, 1977; Dunlop et al., 1978), has two L-asparaginases specified by apnA and Ahr A, respectively, apnA has been mapped to a position on the left arm of linkage group II, in the sequence adH--acrA--apnA--wA--methA--palcA--(centromere). Cell, 1980 Nov, 22(2 Pt 2), 333 - 48 Sequence of introns and flanking exons in wild-type and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron; Lazowska J et al.; We have determined the DNA sequence of the wild type and mutated introns as well as their flanking exons in the yeast mitochondrial gene specifying cytochrome b . The second intron (box3) encodes a trans-acting protein "mRNA maturase" responsible for splicing and maturation of cytochrome b mRNA . This protein is interlaced with cytochrome b exon sequences . Its biosynthesis is subject to a negative feedback which may constitute a regulatory mechanism for the expression of split genes. Eur J Biochem, 1980 Nov, 112(2), 363 - 6 A histone-specific acetyltransferase is associated with simian-virus-40 chromatin; Otto B et al.; {14C}Thymidine-labeled simian virus 40 nucleoprotein complexes were prepared from nuclei of lytically infected African green monkey kidney cells . The nucleoprotein complexes were further purified by sucrose gradient centrifugation and then incubated in the presence of {3H}acetyl-coenzyme A . We observed a transfer of {3H}acetyl groups to the endogenous histones H2B, H3 and H4 . The enzyme was solubilized in the presence of 0.5 M NaCl and showed properties described for the DNA-binding acetyltransferase (J . Bohm, E . J . Schlaeger and R . Knippers, preceding paper in this journal). Biochemistry, 1980 Oct 28, 19(22), 4993 - 9 Inhibition of orotidine-5'-phosphate decarboxylase by 1-(5'-phospho-beta-d-ribofuranosyl)barbituric acid, 6-azauridine 5'-phosphate, and uridine 5'-phosphate; Levine HL et al.; 1-(5'-Phospho-beta-D-ribofuranosyl)barbituric acid, an analogue of orotidylic acid, binds to orotidine-5'-phosphate decarboxylase about 100000 times as strongly as does the substrate . The Ki at pH 6 is 9 X 10(-12) M and the half-time for dissociation at 4 degrees C is about 10 h . The binding of the barbiturate analogue to the enzyme is thus one of the strongest interactions between small molecules and proteins that have been measured . The possibility that the inhibitor is a transition-state analogue is discussed. Biochem J, 1980 Oct 1, 191(1), 247 - 51 Triazine dyes, a new class of affinity labels for nucleotide-dependent enzymes; Clonis YD et al.; A number of reactive dichlorotriazine dyes specifically and irreversibly inactivate pig heart lactate dehydrogenase, yeast glucose 6-phosphate dehydrogenase and yeast hexokinase at sites competitive with NAD+, NADP+, and ATP respectively . Monochlorotriazine dyes, including Cibacron Blue F3G-A, do not inactivate lactate dehydrogenase but display high affinity and thus inhibit the inactivation by dichlorotriazine dyes . These data are interpreted in terms of the ability of nucleotide-binding enzymes to bind polysulphonated aromatic chromophores. Biochem J, 1980 Oct 1, 191(1), 269 - 72 Ready separation of proteins from nucleoprotein complexes by reversible modification of lysine residues; Shetty JK et al.; Modification of proteins with citraconic anhydride altered the electrostatic relationship between cationic epsilon-NH3+ groups of lysine residues of proteins and anionic phosphate groups of nucleic acids, thereby destabilizing the nucleoprotein complex . This procedure facilitated the separation of proteins from nucleic acids at pH4-4.2 . The modifying groups were then deacylated from the proteins under acidic conditions (pH3-6) at 30 degrees C. J Biol Chem, 1980 Sep 25, 255(18), 8756 - 60 31P NMR quantitation of the displacement of equilibria of arginine, creatine, pyruvate, and 3-P-glycerate kinase reactions by substitution of sulfur for oxygen in the beta phosphate of ATP; Lerman CL et al.; 31P NMR measurements have been found to be a convenient means for simultaneously measuring the concentrations of several species in the equilibrium mixtures of the reactions catalyzed by arginine kinase and creatine kinase . MgATP + X in equilibrium MgADP + XP where X = arginine or creatine and XP = P-arginine or P-creatine . The free energy of phosphorylaton of various metabolites by adenosine 5'-O-(2-thiotriphosphate) at pH 8.0 and 30 degrees C is more exergonic than the corresponding phosphorylations by ATP by about 2.5 kcal/mol, resulting in a displacement of the equilibrium toward the nucleoside diphosphates by a factor of approximately 60 . Since this factor does not depend on the nature of the metabolite, the equilibrium constants of thionucleotide reactions may be used to determined the equilibrium constants of corresponding oxynucleotide reactions which lie too far toward ATP . The equilibrium constants of the oxynucleotide reactions catalyzed by pyruvate kinase and 3-P-glycerate kinase calculated by this method from the experimentally determined equilibrium constants of the corresponding thionucleotide reactions are 3.1 x 10(-4) and 2.9 x 10(-4), respectively, under the experimental conditions used . The equilibrium constants and degree of stereoselectivity of the arginine kinase reaction are altered when Ca2+ replaces Mg2+ as the activating metal ion. Hoppe Seylers Z Physiol Chem, 1980 Sep, 361(9), 1363 - 9 {An improved rotational correlation method for the structure determination of biological macromolecules by averaging of electron micrographs (author's transl)}; Steinkilberg M et al.; The averaging of electron micrographs of biological macromolecules by correlation techniques can be performed if the molecules lie plane to the support film . To improve the normal method which uses the autocorrelation function to determine the orientation of the molecules, a new method and its computer realization has been devised using iterative, translational and rotational correlations of the molecule images themselves . With this method the symmetry properties of yeast fatty acid synthetase have been studied . The results show: 1) The new method is very precise (angle accuracy ca . +/- 1 degrees) . 2) Fatty acid synthetase contains a threefold rotation axis . A cyclic symmetry (C3) of the structure is likely . 3) The alpha-subunit consists of at least 2 protein domains. Gut, 1980 Aug, 21(8), 689 - 94 Studies on long chain fatty acid:CoA ligase from human small intestine; Bierbach H; Long chain fatty acid:CoA ligase (EC 6.2.1.3.) was examined in human small intestinal mucosa using the hydroxamate-trapping method . With optimal assay requirements using palmitate as substrate a significant difference of specific activities could be detected in the total homogenate from duodenum, 40.9 +/- 11.6 nmol/min per mg protein versus upper jejunum, 51.9 +/- 13.7 (P less than 0.05) . The enzyme activity of the microsomal fraction of upper jejunum was 101.8 +/- 44 nmol/min per mg protein . ATP, CoA, and Mg2+ were essential constituents of the reaction . A broad pH-optimum was observed between 6.75 and 7.75 with a maximum at a pH of 7.25 . Whereas palmitate in the presence of albumin revealed a wide range of optimal concentration in supporting maximal enzyme activity, oleate was found to strongly inhibit the reaction . Where substrate specificity with both the total homogenate and the microsomal fraction was concerned, maximal reaction rates were obtained with palmitate for the long chain saturated fatty acids C12:0' C14:0' C16:0' and C18:0' and with oleate for the long chain unsaturated fatty acids C18:1 C18:2' and C18:3' respectively . The highest specific activity of the enzyme was localised in the microsomal fraction . The kinetic data and properties of the long chain fatty acid: CoA ligase from human intestine are discussed with respect to the intestinal enzyme from other species. Fed Proc, 1980 Aug, 39(10), 2736 - 9 Advances in our understanding of vitamin E; Scott ML; Vitamin E is an essential nutrient for animals and man since it is not synthesized in the body . The level of vitamin E in the lipoproteins of plasma and in the phospholipids of vital mitochondria, microsomes, and plasma membranes in humans depends (as in experimental animals) on the amount of biologically active vitamin E being consumed, the levels of dietary prooxidants and antioxidants, and the adequacy of dietary selenium . Studies with chicks have demonstrated an important mode of action of both vitamin E and selenium in metabolism, and provide a solution to the long-term enigma of the true role of vitamin E in the diet of all animals, including man . The biochemical actions of vitamin E and selenium are concerned with prevention of peroxidative damage to cells and subcellular elements, thereby aiding the body in maintaining its normal defense mechanisms against disease and environmental insult. Proc Natl Acad Sci U S A, 1980 Aug, 77(8), 4679 - 82 Chemical probes for higher-order structure in RNA; Peattie DA et al.; Three chemical reactions can probe the secondary and tertiary interactions of RNA molecules in solution . Dimethyl sulfate monitors the N-7 of guanosines and senses tertiary interactions there, diethyl pyrocarbonate detects stacking of adenosines, and an alternate dimethyl sulfate reaction examines the N-3 of cytidines and thus probes base pairing . The reactions work between 0 degrees C and 90 degrees C and at pH 4.5--8.5 in a variety of buffers . As an example we follow the progressive denaturation of yeast tRNAPhe terminally labeled with 32P as the tertiary and secondary structures sequentially melt out . A single autoradiograph of a terminally labeled molecule locates regions of higher-order structure and identifies the bases involved. Nature, 1980 Jul 24, 286(5771), 352 - 6 Insertion of the eukaryotic transposable element Ty1 creates a 5-base pair duplication; Farabaugh PJ et al.; The his4-912 mutation results from insertion of a transposable element into the 5'-non-coding region of the his4 gene of yeast . A duplication of 5 base pairs of wild-type his4 DNA flanks the inserted element . The major class of His+ revertants result from excision of most of the transposable element, leaving an inserted segment of 334 base pairs flanked by the 5-base pair repeats. J Biol Chem, 1980 Jul 10, 255(13), 6224 - 7 The binding of cytochrome c peroxidase and ferricytochrome c . A spectrophotometric determination of the equilibrium association constant as a function of ionic strength; Erman JE et al.; Complex formation between cytochrome c peroxidase and ferricytochrome c perturbs the optical absorption spectrum in the Soret band by about 2% . This perturbation can be utilized as a measure of the complex formed in solution and permits the determination of the stoichiometry and the equilibrium association constant for this reaction . At pH 6, in cacodylate/KNO3 buffers, only a 1:1 complex between cytochrome c peroxidase and ferricytochrome c is detected . The equilibrium association constant for the complex has been determined as a function of ionic strength and varies between (6.0 +/- 3.6) x 10(6) M-1 and (2.2 +/- 1.9) x 10(6) M-1 over the ionic strength range 0.01 M to 0.20 M. Biochim Biophys Acta, 1980 Jun 27, 608(1), 54 - 61 Initiation of protein synthesis in isolated mitochondria and chloroplasts; Lucchini G et al.; N5-Formyltetrahydrofolate, a competitive inhibitor of the formylation of the initiator Met-tRNAfMet in an in vitro assay, is a powerful inhibitor of amino acid incorporation in isolated Saccharomyces cerevisiae mitochondria and in Euglena gracilis chloroplasts . Thus, a large part of the incorporation is dependent upon new initiation acts . On the contrary, the rate of incorporation can be largely increased by addition of the specific formyl group donor, N10-formyltetrahydrofolate . Experiments are also reported strongly suggesting that the formylation of Met-tRNAfMet is an absolute requirement in order to initiate protein synthesis in chloroplasts, as has been shown in mitochondria. Biochim Biophys Acta, 1980 Jun 13, 613(2), 292 - 308 Study of subunit interactions in immobilized D-glyceraldehyde-3-phosphate dehydrogenase; Cherednikova TV et al.; Under conditions which cause dissociation of soluble tetrameric glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) into inactive dimers, immobilized apoenzymes from yeast and rat skeletal muscle coupled to CnBr-activated Sepharose via one subunit retain 50% of matrix-bound protein with unaltered specific activity . The solubilized dissociated species are inactive . Two molecules of NAD+ (NADH) firmly bound to the immobilized rat muscle tetramer can prevent the dissociation . Immobilized dimer was demonstrated to bind one molecule of coenzyme with high affinity . Using various combinations of immobilized and soluble rat muscle and yeast dimers, we succeeded in reconstituting tetramers, containing one molecule of NAD+ bound either to a matrix-linked or to a non-covalently bound dimer . In the latter case, the dissociation of the tetramer was completely prevented . This suggests that the binding of a single coenzyme molecule is sufficient to stabilize the interdimeric contacts provided the neighbouring dimer is stabilized independently . Such stabilization is produced by the covalent binding of one of the subunits comprising the dimer to the matrix . The structure of the dimer as a whole becomes resistant to the action of the dissociating agent . The effect appears to be cooperative and similar to that of NAD+ or NADH . The dissociation of the immobilized tetramer is, most likely, the result of conformational changes, affecting the structure of the non-covalently bound dimer . Any factor, capable of preventing these changes, would stabilize the interdimeric contacts . The latter conclusion is substantiated by the effect of specific antibodies, which prevent the dissociation of the immobilized tetramer by forming a complex with the dimer, non-covalently bound to the matrix . The evidence obtained in the present investigation supports the conclusion that the isolated dimer of glyceraldehyde-3-phosphate dehydrogenase represents a relatively independent structural and functional 'unit' of the enzyme . It can be stabilized in a catalytically active form by interactions other than those involved in inter-dimeric contacts in the tetramer . The kinetics of the association of immobilized and soluble dimers have been studied . Association rate constants were determined for homologous (yeast-yeast, rat-rat) and heterologous (yeast-rat, yeast-rabbit) dimer combinations . The binding of one molecule of specific antibody to the immobilized dimer was shown to increase the rate constant of association. Biochim Biophys Acta, 1980 Jun 13, 613(2), 275 - 91 Subunit interactions in glyceraldehyde-3-phosphate dehydrogenases . Their involvement in nucleotide binding and cooperativity; Scheek RM et al.; 1 . The hybridization of rabbit muscle and yeast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) was used to study the involvement of subunit interactions in NAD+ and NADH binding by these enzymes . 2 . In the presence of 1 mM NAD+ or NADH no hybrid formation was observed with our preparations of the two enzymes . 3 . The inhibition by NADH of the hybrid formation is shown to be a consequence of an unfavourable equilibrium of the hybridization process in the presence of NADH . 4 . The inhibition by NAD+ of the hybrid formation is shown to be a consequence of both a shift in the equilibrium, as in the case of NADH, and a decrease in the rate of the dissociation of the enzymes . 5 . The dimer of the yeast enzyme binds NAD+ or NADH with equal affinity irrespective of whether it is combined with another yeast dimer in the yeast tetramer or with a rabbit muscle dimer in the hybrid . 6 . The binding of NAD+ and NADH to the dimer of the rabbit muscle enzyme is stronger in the rabbit muscle tetramer than in the hybrid; this explains the shift in the equilibrium of the hybridization process caused by these nucleotides . 7 . Alkylation of the rabbit muscle enzyme with iodoacetate does not influence the hydridization process in the absence of nucleotides . 8 . After alkylation of the rabbit muscle enzyme NADH cannot cause a large shift in the equilibrium of the hybridization process . 9 . In accordance with this it was found that the binding of NADH (and NAD+) to the rabbit muscle enzyme is weakened by alkylation, whereas the binding of NADH to the alkylated rabbit muscle subunits is not affected strongly by the hydridization . 10 . An attempt is made to combine the effects of nucleotides on the hybridization properties of the yeast enzyme and the alkylated or unalkylated rabbit muscle enzymes with the binding properties of all tetrameric species involved in the hybridization processes in a thermodynamic description of nucleotide binding and subunit interactions. Nucleic Acids Res, 1980 Jun 11, 8(11), 2365 - 75 Transcription of killer virion double-stranded RNA in vitro; Welsh D et al.; Intracellular virions of stationary phase ethanol-grown cells of a killer strain of Saccharomyces cerevisiae contain encapsulated M (1.1 x 10(6) dalton) and L (3.2 x 10(6) dalton) double-stranded RNA plasmids . These virions also contain RNA polymerase activity which catalyzes the synthesis of full-length, single-stranded, asymmetric transcripts (denoted m and l) of the virion double-stranded RNAs . Product m is made by M-containing particles and shows complete sequence homology to M but not to L . Product l is made by L-containing particles and shows complete homology only to L . The products show no self-homology, indicating asymmetric transcription . Therefore, the polymerase appears to function in vitro as a double-stranded RNA transcriptase . The lack of sequence homology between M and L is confirmed. In Vitro, 1980 Jun, 16(6), 541 - 6 Mutations and cell transformation with 2-azido-9-fluorenone oxime; Dollinger MD et al.; When photolyzed in situ for as little as 15 s 2-azido-9-fluorenone oxime causes Type II and Type III transformation in C3H 10T 1/2 CL8 cells . In the yeast strain Saccharomyces cerevisiae D-7, simple reversions and gene conversions occurred and also mitotic crossing over, to a lesser extent, but no mitochondril "petite" mutants occurred . No mutations or transformations were induced in the dark or by the light itself. Prikl Biokhim Mikrobiol, 1980 May-Jun, 16(3), 383 - 7 {Preparation and charaterization of aminopolysterol immobilized invertase}; Pauliukonis AB et al.; Invertase from Saccharomyces cerevisiae was immobilized on aminopolysterol by adsorption, glutaraldehyde, carbodiimide or bromacetyl methods . The dependence of activity of invertase immobilized by the carbodiimide method upon pH, temperature and sucrose concentration was studied . The "effective" Michaelis constant of the immobilized preparation was 6 times higher than that of soluble inverase . At high sucrose concentrations (beginning with 0.2 M for immobilized and 0.9 M for soluble invertase) substrate inhibition was observed . The data on stability of immobilized invertase during refrigerated storage and in a working flow column were obtained. J Biomech Eng, 1980 May, 102(2), 91 - 7 Volumetric changes in cells during freezing and thawing; Knox JM et al.; A thermodynamic model is presented to describe the combined freezing and thawing process for living cells . Continuous changes in the cell volume are predicted according to the thermal protocol imposed on the system . Experimental verification of the model is sought by monitoring continuously the volume of cells as frozen on a cryomicroscope . The volumes of individual cells are measured from sequential photomicrographs by a computerized image analysis technique . The model and experimental data are in quite close agreement for the freezing process, but upon thawing the experimentally measured volumes consistently increased much more rapidly than predicted by the model . The model can be made to conform to the data by accounting for a substantial influx of electrolyte to the cell at subfreezing temperatures. Scand J Clin Lab Invest, 1980 May, 40(3), 271 - 8 Intracellular localization of palmitoyl-CoA hydrolase and palmitoyl-CoA synthetase in human blood platelets and liver; Berge RK et al.; The intracellular localization studies of human blood platelets by sucrose gradient and differential centrifugation, showed that palmitoyl-CoA hydrolase was mainly localized in the cytosol fraction . However, a localization also in the mitochondrial fraction seems possible as disruption of platelets by the French press and nitrogen decompression techniques resulted in mitochondrial damage . In human liver the palmitoyl-CoA hydrolase was localized in the mitochondrial and microsomal fractions . Palmitoyl-CoA synthetase was localized in the mitochondrial and microsomal fractions in both platelets and liver . The possible physiological implications of these differences, and the finding of a very high ratio of palmitoyl-CoA hydrolase/palmitoyl-CoA synthetase in platelets compared with liver, are discussed. J Biol Chem, 1980 Apr 25, 255(8), 3240 - 1 Shift of the equilibrium constant of the 3-P-glycerate kinase reaction towards 1,3-bis-P-glycerate with adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) as substrate; Jaffe EK et al.; It has been demonstrated that the reaction of ATP beta S and 3-P-glycerate catalyzed by 3-P-glycerate kinase, unlike the reaction with ATP, can form a readily detectable amount of 1,3-bis-P-glycerate as observed by 31P NMR . By quantifying production of 1,3-bis-P-glycerate from the phosphorothioate analogue of ATP as a function of time as the reaction approaches equilibrium, Keq for the reaction was estimated to be approximately 400, about 1 order of magnitude less than the equilibrium constant previously reported for the analogous reaction of the normal nucleotide substrates. Biochim Biophys Acta, 1980 Mar 26, 622(1), 144 - 50 Enzymatic trimethylation of residue-72 lysine in cytochrome c . Effect on the total structure; Kim CS et al.; A highly purified protein methylase III from Neurospora crassa or Saccharomyces cerevisiae specifically methylates a single lysine residue of position 72 of horse heart cytochrome c . The enzymatically methylated cytochrome c has been separated from the unmethylated counterpart species by isoelectric focusing . Simultaneously, the pI values of these two species were found to be 9.49 and 10.03, respectively . Since methyl substitution increases the basicity associated with the epsilon-amino group of lysine residues, the observed decrease in pI value is in opposition to the predicted increase . Space-filling models revealed the possibility of a hydrogen bond between the oxygen of amide of residue-70 asparagine and the epsilon-amino nitrogen of residue-72 lysine in unmethylated horse heart cytochrome C . the enzymatic methylation of residue-72 lysine tends to dissociate this hydrogen bond, thereby possibly inducing the shift of 'effective charge' of the protein molecule . This paper also deals with the pI values of cytochromes c from 13 different sources, determined by the isoelectric focusing technique. Nucleic Acids Res, 1980 Mar 25, 8(6), 1435 - 42 Nucleotide sequence of the mitochondrial genes coding for tRNAglyGGR and tRNAvalGUR; Miller DL et al.; Yeast mitochondrial DNA-pBR322 recombinant DNA molecules known to contain tRNA genes from a tRNA rich region of the yeast genome were used as a source of DNA for restriction mapping and tRNA gene sequence analysis . We report here restriction maps of two segments of yeast mitochondrial DNA and the sequence of mitochondrial genes coding for tRNAglyGGR and tRNAvalGUR . Both genes are flanked by A + T rich DNA and neither has an intervening sequence nor codes for a 3' CCA end . The tRNA structures deduced from the genes have the usual cloverleaf structures and invariant nucleotides . This combination of DNA sequencing and restriction mapping has enabled us to determine that the tRNAvalGUR and a previously sequenced tRNA, the tRNApheUUY are transcribed from the same strand of DNA. Nature, 1980 Mar 13, 284(5752), 143 - 8 Order and intracellular location of the events involved in the maturation of a spliced tRNA; Melton DA et al.; Microinjected frog oocytes were used to analyse the RNA processing steps which lead to the appearance of a mature cytoplasmic tRNAtyr molecule . The results show that removal of the intervening sequence from within a yeast tRNAtyr precursor, excision of extra 3' and 5' nucleotides, addition of a 3'-terminal CCA and modification of at least seven ribonucleotides all occur in the nucleus before the tRNAtyr is transported to the cytoplasm . Moreover, we find that the ribonucleotide modifications occur in a strict order which precisely correlates with the size alterations of the tRNAtyr precursor. Biokhimiia, 1980 Mar, 45(3), 468 - 73 {Formation of oligomeric form of transketolase and the factors influencing this process}; Solovbeva ON et al.; The immobilized transketolase (dimer) is a mixture of two enzyme forms, i . e . a completely active and completely inactive ones . The decomposition of the active enzyme form into subunits to form immobilized monomers occurs at a higher rate than that of the inactive form . The reassociation of the monomers with a formation of dimers strongly depends on pH, the pH optimum of reassociation being equal to 7,9--9,2 . Thiamine pyrophosphate and Ca2+ as well as Ca2+ alone but taken at sufficiently high concentrations favours the association of the monomers into a dimer . The reassociated dimers exhibit a complete catalytic activity only when the reassociation occurs in the presence of Ca2+ . The activity of reassociated dimers obtained in the absence of Ca2+ was decreased by 50% . The reassociation capacity of the immobilized subunits obtained in the absence of beta-mercaptoethanol was considerably decreased . The immobilized subunits obtained in the presence of beta-mercaptoethanol and then treated by N-ethylmaleimide were not capable of reassociation. Eur J Biochem, 1980 Mar, 104(2), 611 - 6 Regulation of phosphatidylethanolamine methyltransferase level by myo-inositol in Saccaromyces cerevisiae; Yamashita S et al.; 1 . A conditional choline auxotroph was isolated . The growth of this mutant was markedly inhibited by the addition of a low concentration of myo-inositol to the culture medium . The growth inhibition was completely prevented by the addition of choline . 2 . When this mutant cell was grown in the presence of myo-inositol, the intracellular level of phosphatidylethanolamine methyltransferase was very low, whereas in the cell grown in the absence of myo-inositol the enzyme level was normal . The addition of myo-inositol to the cell grown in the absence of myo-inositol caused a marked decrease in the methyltransferase level . 3 . The reduction in phosphatidylethanolamine methyltransferase level by myo-inositol also occurred in various wild type strains of Saccharomyces cerevisiae . 4 . The decreased methyltransferase activity was restored by the removal of myo-inositol from the culture medium . The restoration of the enzyme level was completely abolished by cycloheximide . Thus, it was shown that protein synthesis was involved in the change of phosphatidylethanolamine methyltransferase level by myo-inositol . 5 . These results suggest that the synthesis of phosphatidylcholine from phosphatidylethanolamine by the methylation pathway is regulated by myo-inositol. Ukr Biokhim Zh, 1980 Mar-Apr, 52(2), 164 - 6 {Effect of HCO3- and carbon dioxide at various concentrations on activity of certain enzymes}; Mel'nikhuk DA et al.; The paper deals with the effect of changes in the concentration of carbonic acid in the medium on the reaction rate catalyzed with enzymes of various spectrum of the action . It is shown that the presence of carbonic acid in the medium reaction increases the rate of reactions catalyzed with lactate dehydrogenase of the rabbit liver soluble fraction, with glucose-6-phosphate dehydrogenase from yeast and trypsin . Under the same conditions the reaction rate catalyzed with glucose-6-phosphate dehydrogenase of the rabbit liver soluble fraction and with ATP-citrate (pro-3S)-lyase is considerably decreased . Changes in the carbonic acid concentrations within the physiological limits are found to have no effect on lactate dehydrogenase from the cattle heart and chymotrypsin. J Cell Biol, 1980 Feb, 84(2), 246 - 60 A morphological study of plasma and phagosome membranes during endocytosis in Acanthamoeba; Bowers B; Particle ingestion by Acanthamoeba is rapid . Within 40 s bound particles can be surrounded by pseudopods, brought into the cytoplasm, and released as phagosomes into the cytoplasmic stream . In electron micrographs the phagosome appears as a flasklike invagination of the surface . Separation from the surface occurs by fragmentation of the attenuated "neck+ of the invagination . The separated phagosome membrane has a three- to fourfold greater density of intramembrane particles than the plasma membrane from which it derives . This change is evident within 15 min of ingestion and is detectable while the membrane is still tightly apposed to the particle . There is no direct evidence for the mechanism of this increase; no increase in particle density was seen in the membrane at an early stage in the forming phagosomes still connected to the surface . These morphological observations are consistent with chemical analyses, to be reported in a separate communication, that show that the phagosome membrane has a higher protein to phospholipid ratio and a higher glycosphingolipid content than the plasma membrane . Enlarged phagosomes (presumptive phagolysosomes) show multiple small vesiculations of characteristic morphology . The small vesicles are postulated to be the major route of membrane return to the cell surface. J Biochem (Tokyo), 1980 Feb, 87(2), 549 - 55 Amino acid sequence of a purothionin homolog from barley flour; Ozaki Y et al.; A purothionin homolog was isolated from barley flour and purified by CM-52 column chromatography . It showed potent lethal activity towards brewer's yeast and its complete amino acid sequence was determined to be as follows . Lys-Ser-Cys-Cys-Arg-Ser-Thr-Leu-Gly-Arg-Asn-Cys-Tyr-Asn-Leu-Cys-Arg-Val-Arg-Gly-Ala-Gln-Lys-Leu-Cys-Ala-Gly-Val-Cys-Arg-Cys-Lys-Leu-Thr-Ser-Ser-Gly-Lys-Cys-Pro-Thr-Gly-Phe-Pro-Lys . It thus consists of 45 amino acid residues with 8 cysteines . The number of amino acid residues and the positions of the 8 cysteines are identical with those of wheat purothionins . There is a high degree of homology in the primary structures of these proteins. Biochim Biophys Acta, 1980 Jan 11, 611(1), 35 - 9 Fibrin membrane endowed with biological function . V . Multienzyme complex of uricase, catalase, allantoinase and allantoicase; Okamoto H et al.; The enzymes (uricase (EC 1.7.3.3), allantoinase (EC 3.5.3.4), and allantoicase (EC 3.5.2.5) which participate in degradation of purine bases, were embedded separately in fibrin membranes formed by fibrinogen-fibrin conversion with thrombin . All of these enzymes together with catalase were also embedded in a single fibrin membrane to make an immobilized multienzyme complex . The multienzyme complex in fibrin membrane thus prepared had an ability of degradation of uric acid to urea and glyoxylic acid via allantoin and allantoic acid . The stability of immobilized uricase or catalase embedded in fibrin membrane upon lyophilization was also tested in a comparison with nonimmobilized enzymes. Blood Cells, 1980, 6(4), 745 - 51 Intracellular red-ox steady states as basis for cell characterization by flow cytofluorometry; Thorell B; The blue fluorescence of intracellular reduced coenzymes {NAD(P)H} can be used for cell characterization by rapid flow cytofluorometry . Different types of cells show different metabolic (red-ox) steady states under given conditions . Examples are given for yeast and isolated rat liver cells. Mol Gen Genet, 1980, 179(1), 169 - 75 A defect in carbon catabolite repression associated with uncontrollable and excessive maltose uptake; Entian KD; The previously isolated recessive mutant allele hex2-3 of Saccharomyces cerevisiae caused a defect in carbon catabolite repression of maltase, invertase, malate dehydrogenase, and respiration but at the same time led to an extreme sensitivity to maltose (Zimmerman and Scheel, 1977; Entian and Zimmermann, 1980) . Addition of maltose to a growing culture of a hex2-3 mutant resulted within 60 to 90 min in an inhibition of growth, glycolysis, and de novo protein synthesis . This was not accompanied by any abnormal levels of glycolysis metabolites or glycolytic enzyme activities . However, inhibitory effects coincided with a dramatic increase in intracellular glucose up to 150 mM relative to cell water as opposed to 2.5 mM in wild-type cells . This abnormal behavior is interpreted as a result of an uncontrolled maltose uptake in hex2 mutants, which in combination with increasing maltase activity results in an accumulation of intracellular glucose . Obviously the amount of available glucose surpassed glycolytic capacity in hex2 mutants . Properties of mutant alleles hex2 and hex1 (see Entian and Zimmermann, 1980) clearly show, that specific gene functions are involved in adapting the rate of sugar uptake into the cell to the actual glycolytic capacity. Mol Biol Biochem Biophys, 1980, 32, 368 - 75 Fluorescent tRNA derivatives and ribosome recognition; Wintermeyer W et al.; The use of fluorescent derivatives of tRNAPhe (yeast) in studies on tRNA conformation and on tRNA-ribosome recognition is described . Evidence is presented which indicates that under physiological conditions with respect to ionic strength and Mg2+ concentration, tRNAPhe exists in at least two conformations . The functional significance of this behavior is discussed on the basis of aminoacylation experiments . The investigation of the ribosome complexes of tRNAPhe labeled in the anticodon and D-loops has provided evidence suggesting that the presence of the codon, although not appreciably altering the apparent association constant, leads to qualitatively different complexes in which the tRNA appears to be rigidly bound to the codon even in the P-tRNA to the ribosome occurs in several steps, which take place only in the presence of the proper codon . One or more of these steps may represent codon-induced conformational changes of the tRNA molecule, which constitute the molecular basis of the highly specific binding of the tRNA to the ribosome. Mol Gen Genet, 1980, 180(1), 99 - 105 The trans action of HMRa in mating type interconversion; Rine J et al.; HML and HMR are the sites of cryptic mating type genes in the yeast Saccharomyces cerevisiae . In the presence of the HO gene, the information from HML or HMR (an a or alpha cassette) is transferred to the mating type locus (MAT) . HML, HMR, and MAT are located on chromosome III, yet are widely separated . Similarly, in other yeasts, at least some of the genes involved in mating typing interconversion are linked to the mating type locus . We demonstrate here that a cassette donor (HMR) and the cassette target (MAT) need not be physically linked for successful mating type interconversion . In particular, we show that HMRa on one chromosome can donate an a cassette to the mating type locus on a homologous chromosome III. Mol Gen Genet, 1980, 180(1), 227 - 9 Genetics of oxidative phosphorylation: allelism studies of mitochondrial loci in the PHO1--OLI2 region of the genome; Darlison MG et al.; In a search for new aerobic-growth deficiency mutations affecting mitochondrial energy-conservation two mit- mutations, namely pho-8 and pho-9, have been isolated . The two mutations are allelic with each other, but not allelic with the previously known pho1 mutations although close linkage is indicated . Allelism studies define three distinct PHO loci clustered in this region which also includes the drug-resistance loci OSS1, OLI2 and OLI4 . The existence of phenotypically-distinct markers makes the region amenable to fine-structure mapping. Mol Gen Genet, 1980, 179(3), 469 - 82 Long range control circuits within mitochondria and between nucleus and mitochondria . I . Methodology and phenomenology of suppressors; Dujardin G et al.; To uncover the functional circuitry both within the mitochondrial genome and between the mitochondrial and the nuclear genome, we have developed a general method for selecting and characterizing genetically suppressor mutations that restore the respiratory capacity of mit- mitochondrial mutants . Several hundreds of pseudo-wild type revertants due to a second unlinked mutation which suppresses a target mit- mutation were isolated . The suppressor mutations were found located either in the nuclear (abbreviated NAM for 'nuclear accommodation of mitochondria') or in the mitochondrial genome (abbreviated MIM for 'mitochondrial-mitochondrial interaction') . The specificity of action of various suppressors upon some 250 different mit- mutations located in several genes was tested . According to this specificity of action, suppressors were subdivided into two major classes: allele specific or gene specific suppressors . Because the cob-box mitochondrial gene has a mosaic organization, we were able to find a novel third class of extragenic suppressors specific for mit- mutations within the introns of this gene . Four examples of suppressors showing various specificities of action illustrate our approach . (1) a nuclear gene controlling specific alleles of different mitochondrial genes; (2) a nuclear gene controlling selectively one intron of a split mitochondrial gene; (3) a mitochondrial gene controlling specific alleles of different mitochondrial genes; (4) a region in one complex mitochondrial gene which controls selectively one intron of another split mitochondrial gene . Different mechanisms of suppression are discussed stressing the alleviation of splicing deficiencies of intron mutations. Bull Cancer, 1980, 67(3), 245 - 54 {Rad-equivalences for mono-and bifunctional furocoumarins (author's transl)}; Averbeck D; Rad-equivalences were established for four mono- and bifunctional furocoumarins, two of them of therapeutical interest . Using an eucaryotic cell system (Saccharomyces cerevisiae) rad-equivalences were determined for lethal effects, the induction of mutations and of intra- and intergenic recombination . For each furocoumarin the rad-equivalences for the genetic effects lay in the same range except those for the induction of intergenic recombination which gave higher values . Based on data obtained on mammalian cells in culture a rad-equivalence could be estimated for the phototoxic effect (cell killing effect) of 8-methoxypsoralen used in PUVA-therapy. Eur J Biochem, 1980, 107(1), 73 - 7 Production of glycolate by oxidation of the 1,2-dihydroxyethyl-thamin-diphosphate intermediate of transketolase with hexacyanoferrate(III) or H2O2; Christen P et al.; In the presence of hexacyanoferrate(III), or other suitable oxidants, transketolase catalyzes the oxidative cleavage of its donor substrates xylulose 5-phosphate or fructose 6-phosphate into glycolate and glyceraldehyde 3-phosphate or erythrose 4-phosphate, respectively . Two moles of hexacyanoferrate(III) are reduced per mole of oxidatively cleaved donor substrate . In analogy to the oxidative trapping of carbanion intermediates of other enzymes {Healy, M . J . & Christen, P . (1973) Biochemistry, 12, 35-41}, the kinetic features of the reaction indicate that the 1,2-dihydroxyethylthiamin diphosphate intermediate is the oxidation-susceptible species . The molecular activity for the oxidative cleavage of fructose 6-phosphate at a hexacyanoferrate(III) concentration of 0.5 mM is 0.2% of that for the normal transfer reaction with erythrose 4-phosphate as acceptor substrate . Glycolate is also produced with H2O2 as oxidant; however, the reaction is at least two orders of magnitude slower than with hexacyanoferrate(III). Scand J Rheumatol Suppl, 1980, 31, 57 - 65 Effects of cryoglobulins and aggregated IGG on in vitro monocyte phagocytosis; Svensson B et al.; Monocyte yeast cell phagocytosis (YCP) was low in presence of cryoglobulins(CG) from 7 of 7 patients with SLE and RA with vasculitis and from 2 of 5 patients with essential cryoglobulinaemia . IgM and C3 were detected only in CG associated with low YCP . Furthermore, low YCP was only found with CG isolated from hypocomplementaemic sera . The results also showed low YCP when heat-aggregated IgG (HAG) greater than or equal to 19S was added to the culture medium . In the presence of HAG or SLE-sera, preopsonized yeast cells could be adequately phagocytosed by monocytes whereas, in parallel cultures, the phagocytosis of untreated yeast cells was low . It is possible that CG and HAG impair YCP in the same way as SLE-sera . The nature of this presumed common mechanism has not been clarified in this limited study . The findings are, however, compatible with the possibility that immune complexes may interfere with the opsonization of the yeast cells, possibly through inactivation of complement components. Scand J Rheumatol Suppl, 1980, 31, 29 - 41 Monocyte in vitro function in systemic lupus erythematosus (SLE) . I . A clinical and immunological study; Svensson B; A previous article (34) has shown that normal monocytes cultivated in presence of several sera from patients with SLE displayed low yeast cell phagocytosis . In the present extended study it was investigated whether the level of phagocytic activity (PA) had any relation to clinical and laboratory expressions of disease activity . The results reveal that, in addition to hypocomplementaemia and cryoglobulinaemia, low PA was associated with high clinical disease activity including arthritis, rash and nephritis and high titres for anti-nDNA . It was also found that PA improved as the disease activity declined . These findings suggest that this in vitro phagocytosis test might be used as an indirect method for detecting clinically relevant circulating immune complexes in SLE . Furthermore, it might be used as a guide in the evaluation of disease activity in severe SLE . The mechanism behind impaired phagocytosis was not elucidated in this study . Inadequate opsonization of the yeast cells or blocking of the monocyte surface receptors by immune complexes were suggested as possible explanations. Acta Biochim Pol, 1980, 27(3-4), 395 - 403 Effect of regulatory mutations of sulphur metabolism on the levels of cysteine- and homocysteine-synthesizing enzymes in Neurospora crassa; Piotrowska M et al.; 1 . Regulation of four enzymes involved in cysteine and homocysteine synthesis, i.e . cysteine synthase (EC 4.2.99.8), homocysteine synthase (EC 4.1.99.10), cystathionine beta-synthase (EC 2.1.22) and gamma-cystathionase (EC 4.4.1.1) was studied in the wild type and sulphur regulatory mutants of Neurospora crassa . 2 . Homocysteine synthase and cystathionine beta-synthase were found to be regulatory enzymes but only the former is under control of the cys-3 - scon system regulating several enzymes of sulphur metabolism, including gamma-cystathionase . 3 . The results obtained with the mutants strongly suggest that homocysteine synthase plays a physiological role as an enzyme of the alternative pathway of methionine synthesis . Cysteine synthase activity was similar in all strains examined irrespective of growth conditions . 4 . The sconc strain with derepressed enzymes of sulphur metabolism showed an increased pool of sulphur amino acids, except for methionine . Particularly characteristic for this pool is a high content of hypotaurine, a product of cysteine catabolism. Mol Gen Genet, 1980, 179(1), 141 - 6 Localization of mucidin-resistant locus muc3 on mitochondrial DNA with respect to ubiquinol-cytochrome c reductase deficient box loci . Locus muc3 is allelic to box2; Takacsova G et al.; Genetic relations between mitochondrial mucidin-resistant locus muc3 and ubiquinol-cytochrome c reductase-deficient box loci have been studied by recombination and petite deletion analysis . It was found that the locus muc3 maps in the segment of mitochondrial DNA corresponding to the locus box2 . The results suggest the participation of box2/muc3 locus in the sequences of the structural gene for cytochrome b. Prep Biochem, 1980, 10(3), 359 - 68 Preparation of oligonucleotides and their use in molecular weight estimations; Lindblow-Kull C et al.; Yeast RNA was used to prepare oligonucleotides employed to calibrate a G-50 Sephadex column . The oligonucleotides' preparation, isolation, desalting and characterization is described . Data obtained by chromatography of the oligonucleotides demonstrate that the molecular weights of oligonoucleotides can be easily determined by interpolation using plots of elution volumes (Ve) versus molecular weights (M) . Errors greater than 20% are obtained if the conventional plot of Ve-Vo/Vs versus log M is used (where Vo is the void volume of the column and Vs is the volume of the column occupied by the inert phase, the G-50 Sephadex). Nucleic Acids Res, 1979 Dec 20, 7(8), 2469 - 82 An improved rapid enzymatic method of RNA sequencing using chemical modification; Mazo AM et al.; A version of rapid gel sequencing procedure based on the analysis of partial endonuclease hydrolizates of chemically modified 5'-32P-labelled RNA is suggested . Complete and selective modification of cytidilic residues by a methoxyamine-bisulfite mixture leads to the unfolding of the RNA secondary structure and, due to this effect, to the generation of a more uniform set of fragments after partial RNAase hydrolysis . The position of cytidines in an RNA sequence can be determined by restricting the hydrolysis of phosphodiester bonds between the modified CMP residues and their 3'-neighbours with T2 and A RNAases . The method was verified with tRNATrp (yeast) and 5S RNA (rat liver and yeast). Mol Cell Biochem, 1979 Dec 14, 28(1-3), 169 - 84 Molecular aspects of cytochrome c oxidase: structure and dynamics; Azzi A et al.; In the last few years much attention has been dedicated to the elucidation of some of the molecular aspects of cytochrome c oxidase . It has been shown conclusively that the enzyme from several sources (yeast, Neurospora, heart, liver) contains seven different subunits, which are asymmetrically inserted in the membrane . All of these are in contact with the lipid bilayer (except subunits V and VI) and to a greater or lesser extent with the water phase as well (except for subunit I) . Subunit II of the enzyme appears to be involved in the formation of the binding site of cytochrome c . The location of the redox groups of the enzyme is still a matter of controversy . Their distance from the cytochrome c heme group is approximately 35 A such that electron tunneling appears to be the only possible mechanism for transporting electrons across such a distance . A proton pump appears to be associated with electron transport and approximately one proton is extruded per electron equivalent reducing oxygen via the enzyme . N,N', dicyclohexylcarbodiimide a well-established inhibitor of H+-translocating ATPases inhibits the proton pump and labels specifically subunit III of the enzyme. Biochim Biophys Acta, 1979 Dec 7, 571(2), 218 - 23 The functional identity of the active centres of transketolase; Meshalkina LE et al.; Direct determination of the number of catalytically active molecules of the coenzyme in holotransketolase (sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycoaldehydetransferase, EC 2.2.1.1) has corroborated our previous data indicating that in the native enzyme there are two active centres . They have been provided to be functionally identical . It has been shown that the decrease in the specific activity of transketolase during its storage is due to inactivation of one of the active centres, having a lower affinity for the coenzyme . The second active centre retains thereby its full catalytic activity. Proc Natl Acad Sci U S A, 1979 Dec, 76(12), 6132 - 6 Electron-nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase: identification of the free radical site with a methionyl cluster; Hoffman BM et al.; The results of electron-nuclear double resonance and electron paramagnetic resonance (EPR) studies on the hydrogen peroxide compound of yeast cytochrome c peroxidase are inconsistent with previous proposals for the source of the EPR signal in this compound, in particular with its identification with an aromatic amino acid radical such as would arise by oxidation of a tryptophanyl side chain . The present observations lead us to propose that the EPR signal is associated with a cluster containing at least one methionine and in which proximate side chains share the charge created by loss of one electron. Histochemistry, 1979 Nov, 64(1), 115 - 8 Fluorescent colloidal gold: a cytochemical marker for fluorescent and electron microscopy; Horisberger M et al.; The gold method was further developed for fluorescent microscopy . Gold granules (12 nm in size) were labelled with rhodamine conjugates of Concanavalin A and avidin . The fluorescent markers were used to mark cell wall mannan on the yeast Saccharomyces cerevisiae either by the one-step, or by the two-step method via a biotinyl derivative of ConA . By fluorescence or transmission electron microscopy, the two-step method was found to achieve a higher density of marking. Biochemistry, 1979 Oct 16, 18(21), 4755 - 61 Conformation transitions of a tRNA--aminoacyl-tRNA synthetase complex induced by tRNAs bearing different modifications in the 3' terminus; Krauss G et al.; The influence of modifications of the 3'-terminal adenosine of tRNAPhe (yeast) on the complex formation between this tRNA and phenylalanyl-tRNA synthetase (yeast) has been investigated by using fluorescence titrations and fast kinetic techniques . Subtle changes in the 3' terminus are reflected by distinct alterations in the two-step recognition process which had been demonstrated earlier for the native substrate tRNAPheCCA {Krauss, G., Riesner, D., & Maass, G . (1977) Nucleic Acids Res . 4, 2253--2262} . Binding experiments with tRNAPheCC, tRNAPheCCA-ox-red, tRNAPheCC2'dA, tRNAPheCC3'dA, tRNAPheCC-formycin, and tRNAPheCC-formycin-ox-red confirm that the 3'-terminal adenosine participates in a conformational change of the tRNA--synthetase complex . This is valid in both the absence and presence of phenylalaninyl-5'-AMP, the alkyl analogue of the aminoacyladenylate . As compared to tRNAPheCCA, a slower conformational change is observed with the competitive inhibitor tRNAPheCC-formycin-ox-red . The reaction enthalpy and/or the quench of the Y-base fluorescence that accompany the conformational change are altered upon binding of tRNAPheC2'dA, tRNAPheCC3'dA, and tRNAPheCC-formycin . It is evident that the final adaptation between tRNA and its synthetase in the complex is determined by the chemical nature of the 3'-terminal nucleotide . This is of vital importance for the specificity of the aminoacylation process. Biochem J, 1979 Oct 1, 183(1), 127 - 32 The exchange of histidine C-2 protons in superoxide dismutases . A novel method for assigning histidine-metal ligands in proteins; Cass AE et al.; The rates of exchange of the C-2 protons of histidine residues in copper-zinc superoxide dismutase are substantially decreased by metal ion binding . This observation was used to distinguish between ligand and non ligand histidine residues in bovine and yeast copper-zinc superoxide dismutases; the effect was shown to depend only on metal ion co-ordination and not as a consequence of concomitant changes in protein structure . Selective deuteration of the zinc-only proteins at pH (uncorrected pH-meter reading) 8.2 and 50 degrees C resulted in the distinction between copper and zinc ligand resonances in the 1H n.m.r . spectrum of the enzymes . This method is proposed as a generally applicable technique for identifying histidine residues as ligands in metalloproteins. Mol Gen Genet, 1979 Oct 1, 175(3), 259 - 65 Abolition of the cyclic variations in radiosensitivity during meiosis in a sporulation mutant blocked in premeiotic DNA synthesis; Hottinguer-De Margerie H et al.; The response to ultraviolet light (254 nm) of two sporulation mutants during the meiotic process was compared to that of a wild type diploid strain of Saccharomyces cerevisiae . The cyclic pattern for cell killing and rho- induction characteristic of diploid wild type cells persists in a strain able to perform the premeiotic DNA synthesis but which is blocked in the further steps of meiosis (spo8 DMS1) . On the contrary, these fluctations are abolished in a derived mutant (spo8 dsm1) which is blocked in the premeiotic DNA synthesis . Under these conditions, the response to cell killing can be dissociated from the observed for rho- induction. Br J Dermatol, 1979 Oct, 101(4), 379 - 89 Photochemotherapy (PUVA) of psoriasis using 3-carbethoxypsoralen, a non-carcinogenic compound in mice; Dubertret L et al.; The carcinogenic risk of photochemotherapy (PUVA) with bi-functional furocoumarins such as 8-methoxypsoralen (8-MOP) which form cross-links in cellular DNA has initiated a search for active but less hazardous psoralens . A new compound, 3-carbethoxypsoralen (3-CPs), studied in the yeast Saccharomyces cerevisiae (eukaryote), has been shown to be very photoactive on DNA and to form only mono-additions to DNA . These lesions appear to be more easily repaired than the cross-links induced by 8-MOP . 3-CPs produces less nuclear genetic events such as nuclear mutations and mitotic crossovers, but more cytoplasmic 'petite' mutations (damage to mitochondrial DNA) than 8-MOP . In mice it was demonstrated that after local or intra-peritoneal administration, in contrast to 8-MOP, 3-CPs is non-toxic, non-erythematogenic, and non-carcinogenic . A study of ten psoriatic patients had shown that local applications of 3-CPs plus UV-A exhibit about the same therapeutic activity for the clearing of psoriatic lesions as local treatment with 8-MOP plus UV-A, but without any localized hyperpigmentation. Proc Natl Acad Sci U S A, 1979 Oct, 76(10), 5134 - 7 Interference of nonsense mutations with eukaryotic messenger RNA stability; Losson R et al.; The fine structure map of the yeast URA 3 gene was established by meiotic recombination, and amber nonsense mutations were located at different points on the map . The effect of the length of the labeling time on the specific radioactivity of ura 3 messenger RNA and on its repartition between poly(A)-RNA and RNA not containing poly(A) has been followed in nonsense mutants . Nonsense mutations reduce the messenger level without lowering its instantaneous rate of synthesis . The strength of the reduction depends on the position of the nonsense codon within the locus and concerns essentially the accumulation of polyadenylylated ura 3 mRNA. Proc Natl Acad Sci U S A, 1979 Oct, 76(10), 4951 - 5 Replacement of chromosome segments with altered DNA sequences constructed in vitro; Scherer S et al.; We have developed a method that may be of general application for the stable introduction of foreign sequences or deletions, constructed in vitro, into the chromosomes of Saccharomyces cerevisiae . No vector sequences are present in the final strains . Ability to transform cells with DNA, availability of a single selective marker, and integration of the transforming DNA by homologous recombination into the chromosomes are the requirements of the system . Any isolated gene can be deleted or altered and then be used to replace the wild-type chromosomal copy . An internal deletion mutant of the his3 gene and a transposition of a galactose-inducible region into chromosome XV have been generated by using the ura3 gene as the selective marker. Eur J Biochem, 1979 Oct, 100(1), 295 - 300 Irreversible inactivation of pyruvate decarboxylase in the presence of substrate and an oxidant . An example of paracatalytic enzyme inactivation; Cogoli-Greuter M et al.; Pyruvate decarboxylase from yeast is progressively inactivated in the presence of pyruvate and an extrinsic oxidant such as 2,6-dichloroindophenol or hexacyanoferrate(III) . The inactivation is linked to the oxidation of the hydroxyethylthiamine diphosphate intermediate to acetate . Removal of low-molecular compounds by gel filtration does not reactivate the enzyme . The rate of inactivation obeys saturation kinetics with respect to substrate concentration and is independent of enzyme concentration . In analogy to the paracatalytic inactivation of other enzymes forming oxidizable carbanion intermediates {Christen, P . (1977) Methods Enzymol.46, 48--54}, the oxidation of enzyme-bound hydroxyethylthiamine diphosphate is thought to generate a transiently reactive intermediate which, without being released from the enzyme, covalently modifies a group at or near the active site . Reconstitution experiments indicate that the protein rather than the coenzyme moiety is modified. Eur J Biochem, 1979 Oct, 100(1), 157 - 64 Lack of correlation between affinity of the tRNA for the aminoacyl-tRNA synthetase and aminoacylation capacity as studied with modified tRNAPhe; Renaud M et al.; The interactions of several modified yeast tRNAPhe {tRNAPhe lacking 7-methylguanine; a fragment comprising about 3/4 of the whole molecule: tRNAPhe (18--76); tRNAPhe (18--76) lacking 7-methylguanine} with yeast phenylalanyl-tRNA synthetase were studied . Upon excision of the 5'-quarter of the tRNAPhe molecule, the residual fragment still tightly binds to the synthetase, but can no longer by aminoacylated . Surprisingly, upon removal of the 7-methylguanine base at position 46 in this fragment, althought the affinity drops by a factor 10, a significant aminoacylation is restored . These results are discussed in terms of molecular flexibility and a model is proposed for tRNA-enzyme interaction, involving multisite recognition. Biochim Biophys Acta, 1979 Sep 27, 564(2), 355 - 7 Mitochondrial DNA from various organisms does not contain internally methylated cytosine in -CCGG- sequences; Groot GS et al.; Mitochondrial DNAs from yeast, Neurospora, rat and calf do not contain internally methylated cytosine in -CCGG- sequences. Nucleic Acids Res, 1979 Sep 11, 7(1), 179 - 92 Site specific enzymatic cleavage of RNA; Donis-Keller H; The hybridization of a DNA oligonucleotide a specific tetramer or longer) will direct a cleavage by RNase H (EC 3.1.4.34) to a specific site in RNA . The resulting fragments can then be labeled at their 5' or 3' ends, purified, and sequenced directly . This procedure is demonstrated with two RNA molecules of known sequence: 5.8S rRNA from yeast (158 nucleotides) and satellite tobacco necrosis virus (STNV) RNA (1240 nucleotides). Proc Natl Acad Sci U S A, 1979 Sep, 76(9), 4365 - 9 Energy-dependent processing of cytoplasmically made precursors to mitochondrial proteins; Nelson N et al.; Earlier work has shown that mitochondrial proteins synthesized in the cytosol are initially made as larger precursors which are then transferred into the organelles and processed to their mature size in the absence of protein synthesis . It is now demonstrated that depletion of the mitochondrial matrix ATP in intact yeast spheroplasts by various combinations of inhibitors and mutations prevents the processing of precursors to the three largest subunits of the mitochondrial F1-ATPase and two subunits of the cytochrome bc1 complex . These polypeptides are all synthesized outside the mitochondria and transported to the mitochondrial matrix or inserted into the mitochondrial inner membrane . In contrast, depletion of the matrix ATP does not inhibit processing of the precursor to cytochrome c peroxidase; this enzyme is located in the mitochondrial intermembrane space which is freely accessible to ATP made in the cytosol . The processing of extramitochondrially made precursors or the transfer of these precursors across the mitochondrial inner membrane is thus dependent on ATP. Biochim Biophys Acta, 1979 Aug 28, 579(2), 253 - 68 Cyanide reactivity of cytochrome c derivatives; Dyer C et al.; The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility . The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome . Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide . The derivative demonstrate markedly different kinetics which indicate the following conclusions . (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction . (2) Compared to native cytochromes, reaction rates are 10-20 fold greater . This is in accord with a more accessible heme crevice, but not a completely opened crevice . For the completely opened case, rate increases are expected to be between three and five orders of magnitude . (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation . A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step . This conformation change has a limiting rate of 6 . 10(-2) s-1. J Biol Chem, 1979 Aug 25, 254(16), 7986 - 98 Quantitative analysis of two-dimensional electrophoretograms; Bossinger J et al.; A method for quantitative analysis of complex film density distributions in autoradiograms is described . The method is intended particularly for measuring the distribution of radioactivity among the proteins resolved by two-dimensional gel electrophoresis but should, of course, be suited to analyzing other two dimensional separations . The film density distribution is first digitized by a high speed rotating drum scanner to generate the image data array that is stored on a magnetic disk . Subsequent analysis involves: 1) data averaging, 2) detection of contours and of their locations, 3) splitting of overlapping spots, 4) conversion of film density to radioactive intensity by means of calibration films, and 5) differentiation and integration to measure the total radioactivity contained in the protein which generates a spot in the autoradiogram . The product of the analysis is a numbered contour map and a table listing coordinates and radioactivity content of each resolved spot . Coordinate transformations for comparison and matching of autoradiograms are also described . A set of utility programs print and graph the data at intermediate stages of the analysis in order to facilitate the checking of procedures and programs. J Biol Chem, 1979 Aug 25, 254(16), 7468 - 71 Transport of proteins across the mitochondrial outer membrane . A precursor form of the cytoplasmically made intermembrane enzyme cytochrome c peroxidase; Maccecchini ML et al.; Cytochrome c peroxidase, a cytoplasmically made enzyme located between the inner and outer membrane of yeast mitochondria, is synthesized as larger precursor in a reticulocyte cell-free lysate as well as in pulsed yeast spheroplasts . When the pulsed spheroplasts are chased, the precursor is converted to the mature apoprotein . When the in vitro synthesized precursor is incubated with isolated yeast mitochondria in the absence of protein synthesis, it is cleaved to the mature form; the mature form co-sediments with the mitochondria and is resistant to externally added proteases . These results, in conjunction with those reported earlier (Maccecchini, M.-L., Rudin, Y., Blobel, G., and Schatz, G . (1979) Proc . Natl . Acad . Sci . U.S.A . 76, 343-347) suggest that the mechanism of protein transport into the mitochondrial intermembrane space is quite similar to that of protein transport into the matrix or the inner membrane. Mol Gen Genet, 1979 Aug, 175(1), 57 - 65 Characterization of the mutator mutation mut5-1; Morrison DP et al.; The mutator mutation mut5-1 has been characterized with respect to a range of parameters which have been used to describe DNA repair mutants of yeast . No marked effect of the mutation on UV-mutability at lower doses was apparent . Diploids homozygous for the mutation are deficient in UV-induced recombination between the alleles his1-1 and hist1-315, mutation being sufficient to account for all the UV-induced histidine prototrophs . Complementation and mapping studies indicate that mut5-1 is allelic to rad51-1, supporting the conclusion of Hastings et al . (1976) that a mutator may increase spontaneous mutation by modifying repair parameters . Both mut5-1 homozygous and heterozygous diploids give rise to spontaneous or UV-induced segregants which appear to be the products of nondisjunction events . The levels of parameiotic recombination (see Sherman and Roman, 1963; Esposito and Esposito, 1974), sporulation and spore viability observed in mut5-1/mut5-1 diploids indicate that the function encoded by RAD51 is required at 2 times during meiosis . An essential role of the function encoded by RAD51 in mitotic and meiotic recombination is indicated. Proc Natl Acad Sci U S A, 1979 Aug, 76(8), 3865 - 9 Protein influence on the heme in cytochrome c: evidence from Raman difference spectroscopy; Shelnutt JA et al.; Raman difference spectra have been obtained for the cytochromes c of a number of species by simultaneous data acquisition from two samples . Frequency differences as small as 0.1 cm-1 can be measured reproducibly by the technique we have developed . In comparisons between cytochromes c isolated from two different species, the frequency differences in the heme vibrational modes range from 0 to 6 cm-1 . The vibrational frequencies of the heme are sensitive to the electronic charge density on the porphyrin macrocycle . The frequency differences are interpreted in terms of the influence of the heme-packed aromatic and highly electronegative amino acid side chains on the pi* charge density and distribution on the heme . Such a control of the electronic properties of the heme by the protein may be important for the function of cytochrome c. J Biol Chem, 1979 Jul 10, 254(13), 5991 - 6 Cytochrome c peroxidase . Interconversion of chemically and enzymatically reactive and unreactive forms of the ferric protein; Mathews RA et al.; Ferric yeast cytochrome c peroxidase in the presence of different anions may assume a number of forms which differ in optical spectra and chemical properties . In solutions whose only anion is acetate, two spectral forms are present together in an equilibrium . Each of these spectral species is believed to bear bound acetate anion . A form characterized by an intense absorption maximum at 620 nm is unreactive enzymatically and does not react with hydrogen peroxide or with dithionite . A form characterized by a less intense absorption near 645 nm is enzymatically and chemically reactive . Increasing temperature and increasing pH displace the equilibrium toward the 645 nm form . Increasing cytochrome c peroxidase concentration favors the 620 nm form . In kinetic experiments in which the 645 nm form is removed by rapid reaction with H2O2 or dithionite, the 620 nm form is converted in a first order reaction (k = 0.36 s-1, 15 degrees C) to the 645 nm form . In solutions whose sole anion is phosphate a 645 nm form is the only demonstrable spectral species . The enzymatic activity and rates of chemical reaction of 645 nm spectral forms occurring in acetate and in phosphate buffers are the same. Biochim Biophys Acta, 1979 Jul 10, 547(1), 161 - 9 Lipid protein interactions in mitochondria . VII . A comparison of the effects of lipid removal and lipid perturbation of the kinetic properties of mitochondrial ATPase; Parenti-Castelli G et al.; We investigated the kinetics of mitochondrial ATPase in bovine heart mitochondria and submitochondrial particles upon treatment with phospholipase A2, or upon addition of n-butanol to perturb the lipid protein interactions . The changes observed are the following: (1) Lipid removal or perturbation with butanol is accompanied by loss of ATPase activity with decrease of both V and of the KM for ATP . (2) There are changes of activation energy of ATPase activity at temperatures above the discontinuity normally observed for membrane-bound enzymes in mitochondria . In particular, butanol abolishes the discontinuity, and induces a constant activation energy of about 32 kcal/mol in the range 8--37 degrees C . (3) Butanol modifies the pH dependence of ATPase shifting the pH optimum from around 10 to less alkaline values . The optimum for Mg2+ concentrations is increased by the solvent . (4) Treatment with phospholipase A2 results in a removal of oligomycin-sensitive ATPase, whereas butanol addition prevents oligomycin inhibition of ATPase . (5) In beef heart mitochondria, a spin-labelled analog of the inhibitor, dicyclohexyl carbodiimide, did not show any change in environment upon butanol addition, unlike that found in mitochondria from Saccharomyces cerevisiae. Biophys J, 1979 Jul, 27(1), 1 - 14 Magnetic resonance studies of the spatial arrangement of glucose-6-phosphate and chromium (III)-adenosine diphosphate at the catalytic site of hexokinase; Petersen RL et al.; The interaction of CrADP, an exchange-inert paramagnetic analogue of Mg-ADP, with yeast hexokinase has been studied by measuring the effects of CrADP on the longitudinal nuclear relaxation rate (1/T1) of the protons of water and the protons and phosphorus atom of enzyme-bound glucose-6-P . The paramagnetic effect of CrADP on 1/T1 of water protons is enhanced upon complexation with the enzyme . Titrations measuring this paramagnetic effect at several enzyme concentrations in the presence of glucose-6-P yielded a characteristic enhancement factor for 1/T1 of water protons and the dissociation constant of CrADP from the ternary enzyme . ADPCr . glucose-6-P complex . The latter value (2 mM) is similar to that obtained from kinetic inhibition studies (Danenberg and Cleland {1975} . Biochemistry . 14:28) . The presence of glucose-6-P increased the enhancement of the water relaxation rate by enzyme-bound CrADP, suggesting the formation of an enzyme . CrADP . glucose-6-P complex . The existence of such a complex was confirmed by the observation of a paramagnetic effect of enzyme-bound CrADP on the l/T1 of the 31P-nucleus and protons of enzyme-bound glucose-6-P . From the paramagnetic effects of enzyme-bound CrADP on the relaxation rates of the 31P-nucleus and the carbon-bound protons of glucose-6-P in the enzyme . ADPCr . glucose-6-P complex, using the correlation time of approximately 0.7 ns, determined from the magnetic field-dependence of 1/T1 of water protons over the range 24.3-360 MHz, a Cr3+ to phosphorus distance of 6.6 +/- 0.7 A and Cr3+ to alpha- and beta-anomeric proton distances of 8.9 and 9.7 A were calculated . These results imply the absence of a direct coordination of the phosphoryl group of glucose-6-P by the nucleotide-bound metal on hexokinase but indicate van der Waals contact between a phosphoryl oxygen of glucose-6-P and the hydration sphere of the nucleotide-bound metal . The distances are consistent with a model that assumes molecular contact between the phosphorus of glucose-6-P and a beta-phosphoryl oxygen of ADP suggesting an associative phosphoryl transfer . Because after phosphorylation of ADP, the metal ion is coordinated to the transferred phosphoryl group, the overall migration of the phosphoryl group during the phosphoryl transfer is approximately 3.6 A toward the nucleotide-bound metal . Little or no catalysis of phosphoryl transfer from glucose-6-P to alpha, beta-bidentate or beta-monodentate CrADP ( less than or equal to 0.05% of the rate found with MgADP) occurred in the presence of hexokinase, as monitored by glucose formation in a coupled assay system using glucose oxidase and peroxidase . The ability of beta, gamma-bidentate CrATP to act as a substrate (Danenberg and Cleland {1975}. Eur J Biochem, 1979 Jul, 98(1), 9 - 18 Cytochrome c oxidase subunits in nuclear and extranuclear cytochrome-aa3-deficient mutants of Neurospora crassa; Bertrand H et al.; The mitochondria of cytochrome-aa3-deficient Neurospora crassa mutants were screened for the seven polypeptide constiuents of cytochrome c oxidase . The polypeptides of the holoenzyme and the unassembled or partially assembled subunits were detected by sodium dodecyl sulfate/acrylamide gel electrophoresis of immunoprecipitates obtained with antiserum to the holoenzyme as well as to several individual subunits . With respect to the mitochondrially synthesized polypeptides of the oxidase, subunits 1 to 3, the results obtained from the analysis of immunoprecipitates were confirmed through the direct electrophoretic analysis of mitochondrial translation products . The results were as follows . 1 . The mitochondria of the cya-2-8 and cya-3-16 nuclear mutants and the {exn-5} cytoplasmic mutant contained a protein complex immunoprecipitated by anti-holoenzyme antibody and composed of the complete set of the seven cytochrome oxidase polypeptides . Only the oxidase subunits 5 and 6 were immunoprecipitated by anti-holoenzyme antibody from the mitochondria of the cyt-2-1 and 299-1 nuclear mutants, even though at least some of the mitochondrially synthesized polypeptides were detected in both mutants by subunit specific immunoprecipitation . 2 . A 'subunit 1' polypeptide larger than the authentic subunit-1 polypeptide of wild-type cytochrome oxidase was found in the mitochondria from two nuclear mutants, cyt-2-1, and 299-1 and the {mi-3} cytoplasmic mutant . This larger polypeptide may be an unprocessed precursor of the 'mature' subunit 1 protein of the holoenzyme . No changes in the apparent molecular weights were found for the polypeptide subunits of cytochrome oxidase in mitochondria of the {exn-5} cytoplasmic mutant and the cya-2-8 and cya-4-23 nuclear mutants . 3 . A nuclear mutant, 299-1, lacks the mitochondrially synthesized subunit-2 polypeptide of cytochrome oxidase . When cells were labelled in the presence of cycloheximide, the subunit 2 content of mitochondria from mutants {exn-5}, cya-2-8, cya-3-16 and cya-4-23 was lower than in mitochondria from wild-type . This deficiency, however, does not appear to be sufficiently severe to fully account for the lack of cytochrome aa3 in these mutants . The cya-4-23 nuclear mutant either is severely deficient in or lacks cytochrome oxidase subunits 5 and 6 . On the basis of these and previously reported observations, it is proposed that the cytochrome oxidase deficiencies of as many as seven of the eight N . crassa cytochrome-aa3-deficient mutants could be caused by genetically imposed alterations in regulatory systems controlling the production of different components of the enzyme. Biochim Biophys Acta, 1979 Jun 19, 578(2), 462 - 75 Oxidation-reduction reactions of copper-thiolate centres in Cu-thionein; Rupp H et al.; Cu-thionein from yeast was investigated by EPR spectroscopy to probe the oxidation state of copper, and the effects on it of oxidizing and reducing agents . At pH 0.2 the copper was released, but no EPR signal from Cu(II) was observed, unless air was present . Optical experiments did not detect any disulphide groups which might have been formed during anaerobic release of copper . The mercurial, p-hydroxymercuribenzoate caused the release of EPR-detectable copper only under aerobic conditions, and EDTA caused release of Cu(II) on heating . No reduction of the copper-thiolate units in Cu-thionein by ascorbate was detected . Potentiometric titrations with hexachloroiridate(IV) or hexacyanoferrate(III) produced several different Cu(II) EPR signals at various stages of oxidation . The former oxidizing agent required a lower oxidation-reduction potential (+350 mV) to oxidize the copper, than the latter (+410 mV) and neither titration was fully reversible . The EPR signal from Cu(II) oxidized by hexachloroiridate(IV) resembled that produced by p-hydroxy-mercuribenzoate in air, suggesting that the copper was released from its thiolate ligands . It is concluded that the EPR non-detectable copper in the native protein is Cu(I) . Oxidation-reduction of the copper-thiolate clusters of Cu-thionein is proposed to be decisive for controlling storage and transport of cellular copper. Biochim Biophys Acta, 1979 Jun 13, 554(1), 1 - 22 Three dimensional microscopic surface profiles of membranes reconstructed from freeze etching electrol micrographs; Krbecek R et al.; A method of three-dimensional reconstruction of the surface profile of artificial and natural membranes from freeze quenched electron micrographs is presented . The method is based on the analysis of the variation in thickness of platinum layers, deposited under an oblique angle . In essence, it is reminiscent of the method of Eratosthenes to measure the earth's radius . The thickness of etch-like protrusions of membranes could be determined to an accuracy of about 3 A . True distances on curved surfaces rather than projections of distances are obtained . The method has been applied to both model membranes and biological membranes . The essential results are: 1 . Detailed information on the symmetry and the molecular structure of the crystalline phases of dimyristoyl phosphatidylcholine was obtained . The microscopic surface profile of the ripple structure observed between the pretransition and the main transition was analysed . In accordance with a previous model we found that the ripple structure is caused by the spontaneous curvature of the monolayers . The surface profiles of the ripple structure and of the low temperature biaxial phase could be clearly distinguished . 2 . The sizes and shapes of lipid domains formed by both thermically and charge-induced lateral phase separation were determined . This showed that the visual inspection of electron micrographs may lead to a considerable underestimation of the domain size . Conclusions may be drawn concerning the different phases formed upon lateral phase separation . 3 . As a biological example, yeast cell membranes were studied . The method allows one to distinguish between different membrane-bound proteins by measuring the width-to-height ratio of the particles . The deformation of the lipid layer in the environment of the proteins may be determined . This deformation contains information about lipid-mediated long-range interactions between membrane proteins. Biochim Biophys Acta, 1979 Jun 12, 585(2), 293 - 9 Intracellular binding of ethidium studied by photoaffinity labeling in vivo; Fukunaga M et al.; The azide analog of 14C-labeled ethidium bromide was mixed with yeast cells and when photolyzed by visible light, formed covalent complexes with all yeast cell organelles . The 14C counts were found in DNA, RNA and protein of yeast subcellular fractions, illustrating the complexity of binding of a drug which appears highly specific in its actions. Biochemistry, 1979 Jun 12, 18(12), 2471 - 80 Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase; Byers LD et al.; The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate . A variety of phosphonates have been shown to substitute for phosphate in this reaction {Gardner, J . H., & Byers, L . D., (1977) J . Biol . Chem . 252, 5925--5927} . The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Brlnsted coefficient, betaeq, of approximately 1 . This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor . Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction . The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate . The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate . The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable . At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s . Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions . Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors . Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)) . Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme . These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme. Nucleic Acids Res, 1979 Jun 11, 6(7), 2499 - 317 Improved methods for the formation and stabilization of R-loops; Kaback DB et al.; Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented . The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5', 8 trimethylpsoralen . R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature . Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration . In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest. Nucleic Acids Res, 1979 Jun 11, 6(7), 2561 - 7 Nucleotide sequence and conserved features of the 5.8 S rRNA coding region of Neurospora crassa; Selker E et al.; The nucleotide sequence of Neurospora crassa 5.8 S rDNA and adjacent regions has been determined . The deduced 5.8 S rRNA sequence of Neurospora differs from the 5.8 S rRNA sequence of Saccharomyces cerevisiae at 13 of 158 residues . Nine of these differences are clustered in a segment capable of forming a short hairpin secondary structure thought to be involved in the 28 S - 5.8 S rRNA complex . These differences occur in pairs such that the potential secondary structure is preserved. J Biol Chem, 1979 May 10, 254(9), 3439 - 43 A protein inhibitor of the mitochondrial adenosine triphosphatase complex of rat liver . Purification and characterization; Cintron NM et al.; A heat-stable protein has been purified from rat liver mitochondria which inhibits the ATP hydrolytic activity of both the soluble and membrane-bound mitochondrial F1-ATPase . The overall purification is about 2400-fold with the major purification step consisting of Sephadex "affinity" chromatography . The purified rat liver inhibitor is homogeneous as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 12,300 . Amino acid analysis reveals a high content of glutamic acid, lysine, and arginine and the absence of cysteine, proline and methionine . Whether tested with the rat liver or bovine heart ATPase, the liver inhibitor is equally as potent and specific as the heart inhibitor preparation of Pullman and Monroy (Pullman, M.E., and Monroy, G.C . (1963) J . Biol . Chem . 238, 3762-3769) . Although the results presented show that the rat liver ATPase inhibitor resembles closely the ATPase inhibitors from other tissues with respect to specific activity and reaction specificity, it is important to note that the rat liver inhibitor is almost 2000 daltons larger than the bovine heart inhibitor, about 5000 daltons larger than ATPase inhibitors of yeast, and contains significantly more lysine residues than both the bovine heart and yeast inhibitors. Mol Cell Biochem, 1979 May 6, 25(1), 3 - 14 Recent results on how aminoacyl transfer RNA synthetases recognize specific transfer RNAs; Schimmel PR; Aminoacyl tRNA synthetases discriminate between tRNA species by a highly specific mechanism . Physical and chemical studies indicate that the synthetases bind along and around the inside of the three-dimensional L-shaped tRNA structure . Studies of mutant tRNAs that affect synthetase interaction tend to confirm this conclusion . However, in contrast to proteins that recognize a specific block of contiguous nucleotide units (e.g., repressors, restriction enzymes, etc.), synthetases appear to interact with spatially disperse elements of the structure . Available evidence suggests that tRNA binding clefts on various synthetases may be roughly similar, with specificity being achieved by the choice of amino acid residues in a few critical positions in the tRNA binding clefts . With this idea in mind, it should be possible to introduce amino acid substitutions into the binding clefts and thereby change tRNA recognition specificity . This has been attempted (by genetic manipulations) and a mutant alanine tRNA synthetase with altered tRNA recognition has been isolated . This enzyme can attach alanine to isoleucine specific tRNA . When presented with valine specific tRNA, a tRNA similar in some structural features to the isoleucine specific tRNA, or with the structurally quite different tyrosine specific tRNA, no significant aminoacylation occurs . Thus, a precise specificity alteration can occur through mutation; this result supports the idea of similarities in synthetase binding clefts, with specificity being achieved by the positioning of amino acids at critical positions in these clefts . Finally, further data have been obtained on the issue of possible transient covalent bond formation between synthetases and tRNAs, as a critical part of the interaction. J Biochem (Tokyo), 1979 May, 85(5), 1157 - 63 Interaction of mitochondrial aspartate aminotransferase with negatively charged lecithin liposomes; Furuya E et al.; Several kinds of hydrophilic proteins were examined to determine their interaction with artificial liposomes . Mitochondrial aspartate aminotransferase (m-GOT) {EC 2.6.1.1}, as well as cytochrome c, was found to interact strongly with negatively charged liposomes . In each case, an appreciable amount of the protein bound to liposomes remained unreleased after raising the salt concentration in the medium . The m-GOT tightly bound to the liposomes was also found to become latent in its enzymatic activity, and could be reversibly activated by solubilization of the liposomes with detergent . This is also the case for cytochrome c, which ceases to be reducible by external reductant, such as dithionite . Furthermore, the tightly bound m-GOT was not susceptible to the proteolytic action of trypsin, or that of Nagarse . From these observations it can be inferred that these basic proteins interact with acidic liposomes not only electrostatically but also hydrophobically . This kind of hydrophobic interaction was not observed in the combination of positively charged liposomes and acidic proteins, including s-GOT . Mitochondrial GOT was shown to be bound to isolated intact mitochondrial, but the bound enzyme was fully active, in contrast to the case of acidic liposomes . The hydrophobic interaction of water-soluble protein with liposomes is discussed in connection with the penetration of matrix enzyme through mitochondrial membranes. Z Naturforsch {C}, 1979 May-Jun, 34C(5-6), 392 - 6 Drug-protein interaction: 8-methoxy psoralen as photosensitizer of enzymes and amino acids; Veronese FM et al.; For a better insight of the molecular basis of the photobiological effects of furocoumarins, the relevance of proteins oxydation by singlet oxygen produced by these substrates under irradiation with long u . v . light was studied . Complex oligomeric as well as simple monomeric purified enzymes with high or low molecular weight and different properties and simple amino acids were irradiated under oxygen in presence of 8-methoxy-psoralen . The effects on both proteins and amino acids were compared with those obtained under similar conditions with typical photosensitizers as methylene blue and Rose Bengal . The results indicated that the photooxydation of proteins, although possible, appears to play a minor role, if any, in the mechanism of action of furocoumarin. Science, 1979 Apr 27, 204(4391), 375 - 80 Space-filling models of kinase clefts and conformation changes; Anderson CM et al.; Space-filling models of yeast hexokinase, adenylate kinase, and phosphoglycerate kinase drawn by computer clearly portray the bilobal character of these phosphoryl transfer enzymes, and the deep cleft which is formed between the lobes . A dramatic conformational change occurs in hexokinase as glucose binds to the bottom of the cleft, which causes the two lobes of hexokinase to come together . A substrate-induced closing of the active site cleft is postulated to occur in other kinases as well . This change may provide a mechanism by which some of these enzymes reduce their inherent adenosine triphosphatase activity and could be a general requirement of the kinase reaction. J Biol Chem, 1979 Apr 10, 254(7), 2480 - 90 Regulatory interaction between mitochondrial genes . II . Detailed characterization of novel mutants mapping within one cluster in the cob2 region; Hanson DK et al.; These studies describe the properties of three mit- mutants designated EM17, EM25, and PZ1, all mapping at two closely linked sites near one of the boundaries of the region of the mitochondrial genome concerned with the specification of cytochrome b . They all exhibit complex phenotypes affecting cytochrome b, cytochrome aa3, and additional polypeptides not found in the wild type . In the case of EM 17 this complexity can be ascribed to the presence of two mutations induced in the course of the initial mutagenic treatment: one, the cob2 mutation proper, is responsible for the loss of cytochrome b which is replaced by an altered, functionally inactive polypeptide, cytochrome b . This polypeptide can be further modified, or even eliminated, by the controlled introduction of another mutation in the cob1 segment of the cob region . The reduction in cytochrome oxidase subunit I, responsible for the effects on cytochrome aa3 and enzymatic activity in EM17, is due to a second (not mit-) mutation that has been located in the par1-proximal segment of the oxi3 region . This second mutation as well as the cob mutation can be overcome, and the respective aspect of wild type function restored to EM17, by recombination with rho- strains retaining the appropriate segment(s) of the wild type genome . The phenotype of the other two mutants is due to a single mutagenic event . This conclusion is confirmed by their ability to restore wild type functions by reversion . The mutation in EM25 appears to be due to a frameshift, which has led to premature chain termination, producing a polypeptide of Mr = 15,000 related to apocytochrome b . This change is accompanied by a decrease in the amount of subunit I of cytochrome oxidase . Revertants fall into three classes: on galactose two produce a polypeptide indistinguishable from apocytochrome b, but vary in its amount, while the third fails to increase apocytochrome b above mutant levels . Production of subunit I is increased but fails to reach wild type levels . Complete restoration of wild type functions can, however, be obtained by recombination of EM25 with rho- (cob2+) strains . Mutation PZ1 results in a complete absence of any polypeptide related to apocytochrome b and of cytochrome oxidase subunit I . These cells produce a novel polypeptide with a Mr = 45,000 not found in the wild type, and unrelated to all its normal polypeptides . Reversion or recombination with rho- (cob2+) strains results in virtually complete restoration of all wild type functions and the elimination of the novel polypeptide. J Biol Chem, 1979 Apr 10, 254(7), 2471 - 9 Regulatory interactions between mitochondrial genes . I . Genetic and biochemical characterization of some mutant types affecting apocytochr