Curr Genet, 1986, 11(2), 85 - 91 Inducible expression of REP1 causes inducible expression of the 2 micron circle stability system; Jayaram M et al.; The yeast plasmid, 2 micron circle, encodes a stability system consisting of the plasmid replication origin, a cis-active locus designated REP3 and two trans-active functions--the products of the REP1 and REP2 genes . We have constructed 2 micron circle derivatives in which the expression of the REP1 gene is placed under the control of the yeast GAL10 promoter . We show that in such plasmids the stability-system is inducible, being turned off by glucose and turned on by galactose . Further, our results unequivocally demonstrate that, of the two potential in-frame ATG codons at which REP1 translation might initiate (as inferred from the 2 micron circle DNA sequence and from the cap site of the major REP1 transcript), the upstream ATG is dispensable without affecting REP1 function . We also illustrate here a simple and general method for constructing in vivo in yeast 2 micron circle analogs which contain desired alterations within specific regions of the 2 micron circle genome. Curr Genet, 1986, 10(7), 527 - 30 Physical mapping and genome organization of mitochondrial DNA from Candida maltosa; Kunze G et al.; Mitochondrial (mt) DNA of the ascomycetous yeast Candida maltosa was isolated and characterized . The mtDNA is circular and the size estimated from restriction analysis performed with 7 endonucleases was 52 kb pairs . A restriction map was constructed, using the cleavage data of four endonucleases . Using mt genes from Saccharomyces cerevisiae, six structural genes (large rRNA, apocytochrome b, cytochrome c oxidase subunit I and subunit II, ATPase subunit 6 and subunit 9) were located on the C . maltosa chondriome by cross hybridization experiments . The comparison between the mt genomes of C . maltosa and six other yeasts showed differences in the overall genome organization. Curr Genet, 1986, 10(5), 339 - 42 The overproducing CYP1 and the underproducing hap1 mutations are alleles of the same gene which regulates in trans the expression of the structural genes encoding iso-cytochromes c; Verdiere J et al.; The CYP1 gene has previously been identified as coding for a positive trans active factor that activates the expression of CYC1 and CYP3, which are the structural genes for isol1- and iso2-cytochrome c . Two phenotypically distinct classes of CYP1 mutations can be obtained indicating that CYC1 and CYP3 are differentially regulated by the product of CYP1 . The HAP1 gene codes for a product which has previously been proved to be necessary for the expression of the heme dependent CYC1-UAS1 cis regulatory sequence . In this article, we show by complementation and recombination that CYP1 and HAP1 are the same gene, moreover we identify hap1-1 as an iso2-cytochrome c underproducer mutation of the CYP1 gene. Gene, 1986, 44(1), 151 - 8 Direct identification of small sequence changes in chromosomal DNA; Huibregtse JM et al.; Dideoxynucleotide chain termination sequencing has been applied directly to genomic DNA templates by annealing radiolabeled oligodeoxynucleotide primers to unique sites in total yeast DNA and extending with avian myoblastosis virus (AMV) reverse transcriptase . The technique is used here to confirm the introduction of selectively altered tRNA genes into the Saccharomyces cerevisiae genome by gene replacement. Chem Biol Interact, 1985 Dec 31, 56(2-3), 333 - 49 Inhibition of aminoacyl-tRNA synthetases by the mycotoxin patulin; Arafat W et al.; The effect of patulin on tRNA aminoacylation has been determined . This mycotoxin inhibits the aminoacylation process by irreversibly inactivating aminoacyl-tRNA synthetases . At neutral and alkaline pH-values, the inactivation occurs mainly by modification of essential thiol groups of the protein, whereas at acidic pH, where the effect is the most pronounced, the modification of other amino acid residues cannot be excluded. Biochemistry, 1985 Dec 31, 24(27), 8005 - 12 Molecular aspects of functional differences between alcohol and sorbitol dehydrogenases; Eklund H et al.; The amino acid sequence of sheep liver sorbitol dehydrogenase has been fitted to the high-resolution model of the homologous horse liver alcohol dehydrogenase by computer graphics . This has allowed construction of a model of sorbitol dehydrogenase that provides explanations why sorbitol is not a substrate for alcohol dehydrogenase, why ethanol is not a substrate for sorbitol dehydrogenase, and what determines its specificity for polyols . An important feature of the model is that one of the ligands to the active site zinc atom is a glutamic acid residue instead of a cysteine residue, which is the corresponding ligand in the homologous alcohol dehydrogenases . This is one component of the structural change that can be related to the different substrate specificities, showing how altered enzymic activity might be brought about by structural changes of the kind that it is now possible to introduce by site-directed mutagenesis and recombinant DNA techniques. Biochemistry, 1985 Dec 17, 24(26), 7783 - 9 Contribution of water to free energy of hydrolysis of pyrophosphate; de Meis L et al.; The energy of hydrolysis of phosphate compounds varies depending on whether they are in solution or bound to the catalytic site of enzymes . With the purpose of simulating the conditions at the catalytic site, the observed equilibrium constant for pyrophosphate hydrolysis (Kobsd) was measured in aqueous mixtures of dimethyl sulfoxide, ethylene glycol, or polymers of ethylene glycol . The reaction was catalyzed by yeast inorganic pyrophosphatase at 30 degrees C . All the cosolvents used promoted a decrease of Kobsd . Polymers of ethylene glycol were more effective than dimethyl sulfoxide or ethylene glycol in decreasing Kobsd . The higher the molecular weight of the polymer, the lower the value of Kobsd . A decrease in Kobsd from 346 M (delta G degree obsd = -3.5 kcal mol-1) to 0.1 M (delta G degree obsd = 1.3 kcal mol-1) was observed after the addition of 50% (w/v) poly(ethylene glycol) 8000 to a solution containing 0.9 mM MgCl2 and 1 mM Pi at pH 8.0 . The association constants of Pi and pyrophosphate for H+ and Mg2+ were measured in presence of different ethylene glycol concentrations in order to calculate the Keq for hydrolysis of different ionic species of pyrophosphate . A decrease in all the Keq was observed . The results are interpreted according to the concept that the energy of hydrolysis of phosphate compounds depends on the different solvation energies of reactants and products. EMBO J, 1985 Dec 16, 4(13A), 3399 - 405 The B chain of PDGF alone is sufficient for mitogenesis; Kelly JD et al.; The platelet-derived growth factor (PDGF) is a mitogen derived from human platelets consisting of two related polypeptides termed A and B chains . The entire B chain of PDGF is highly (96%) homologous to a portion of p28sis, the transforming protein of simian sarcoma virus . It has been suggested that p28sis exerts its transforming potential by mimicking the growth promoting activity of PDGF and stimulating the cell in an autocrine manner . We have directly examined the mitogenic potential of p28sis and the B chain homologous region by expressing these heterologous sequences in the yeast Saccharomyces cerevisiae . In our constructions, these proteins are encoded by portions of the v-sis gene . Expression and secretion from the yeast cell is achieved by using a yeast promoter and the alpha-factor pheromone secretory leader . The sis proteins thus expressed and secreted are immunoreactive with anti-PDGF antisera and are mitogenic for cultured fibroblasts . Furthermore, they mediate this mitogenic activity by specific binding to the PDGF cell surface receptor . Gel electrophoresis and cell binding analysis indicates that the mitogenic species is primarily a disulphide-bonded dimer . We are able to conclude that p28sis is a mitogen and that a polypeptide corresponding to the B chain alone is sufficient to account for the mitogenic activity attributed to PDGF. Biochem Pharmacol, 1985 Dec 15, 34(24), 4305 - 10 Effects of diabetes mellitus on renal fatty acid activation and desaturation; Clark DL et al.; We report the first direct measurement of delta-6 desaturase and delta-9 desaturase (EC 1.3.99.3, acyl-CoA dehydrogenase) activities in the rat kidney . Crude renal cortical homogenates from alloxan-diabetic and from normal rats were assayed for delta-6 and delta-9 desaturase activities . The delta-6 desaturation pathway activity measured with 9,12-octadecadienoic acid (linoleic acid) as substrate was increased, while the delta-9 desaturation pathway measured with hexadecanoic acid (palmitic acid) as substrate was unchanged in diabetic renal cortex, suggesting that the two enzymes are regulated independently in this tissue . In contrast to the kidney, delta-6 desaturase pathway activity was unchanged and the delta-9 desaturase pathway activity was greatly depressed in diabetic liver . When exogenous long-chain acyl-CoA synthetase (EC 6.2.1.3; acid: CoA ligase, AMP-forming) was added to the delta-6 desaturase assay system, the rate of delta-6 desaturation in normal kidney increased to a rate similar to that found in diabetic kidney; rates in diabetic extracts were unchanged . These results suggest that the rate of fatty acid substrate activation to the coenzyme A ester limits the rate of delta-6 desaturation in normal renal cortex . These results also suggest that the rate of fatty acid activation by long-chain acyl-CoA synthetase activity is increased in diabetic renal cortex . Direct measurement of the activity of long-chain acyl-CoA synthetase demonstrated that its activity was indeed increased significantly in the renal cortex of diabetic rats. Anal Biochem, 1985 Dec, 151(2), 526 - 33 Isolation of specific tRNAs using an ionic-hydrophobic mixed-mode chromatographic matrix; Bischoff R et al.; The coating of a C18-reversed-phase high performance liquid chromatography support (octadecylsilyl-Hypersil) with a tetraalkylammonium salt (methyltrioctylammonium chloride) produces a chromatographic matrix with both ionic and hydrophobic character . Using this material oligonucleotides and tRNAs can be separated with high resolution . The observed resolution is in part due to the apparent lack of diffusion processes occurring during chromatography with this matrix . Some tRNAs can be obtained in high purity from a bulk tRNA mixture after a single chromatographic step . In general it is more efficient to use the matrix as the last step of a purification procedure for a particular tRNA . A two-step procedure is described which allows, in some cases, the isolation of small quantities of specific tRNA isoacceptors. Anal Biochem, 1985 Dec, 151(2), 327 - 33 A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K; Sinha U et al.; Reaction of proteins with Woodward's Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-1 cm-1 . This absorbance can be used to quantitate the reaction of Woodward's Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react . The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1985 Dec, 181(6), 461 - 8 {Effect of lead and cadmium on the viability and phagocytosis of human polymorphonuclear leukocytes}; Baginski B; Studies were performed to test the effects of lead and cadmium on viability and phagocytic activity of human polymorphonuclear leukocytes (PMNL) in vitro . Viability was tested by dye exclusion test and leakage of lactate dehydrogenase (LDH), the phagocytic activity was assessed by counting of ingested yeasts (Saccharomyces cerevisiae) in 100 PMNL . It was demonstrated that the viability of human PMNL was only slightly decreased following 20 h incubation with the metals . This finding is in contrast to findings of studies with macrophages of rodents when viability was rather decreased especially by cadmium . However, both metals were found to markedly suppress phagocytic activity of human PMNL already following 30 min of preincubation with the metal salts . Thus, independent of effects on cell viability the defense mechanisms of human PMNL against infectious agents are markedly reduced in presence of lead or cadmium. Cryobiology, 1985 Dec, 22(6), 537 - 46 Preservation of viable cells in the undercooled state; Mathias SF et al.; Previous studies into the mechanisms governing the freezing of cells in the absence of extracellular ice have been extended to develop a method for the preservation of viable cells in the undercooled state . Deep undercooling of cells is achieved by suspending fine droplets of the cells in oil to make an emulsion, thus minimizing initiation of extracellular ice nucleation . Attempts to preserve yeast cells, cultured sainfoin cells, and dissected shoot-tips (pea and potato) in this way are described . The main findings are that yeast cells can be preserved undercooled at -20 degrees C for at least 16 weeks with no detectable loss of viability, showing that -20 degrees C is a low enough temperature for inhibition of significant biochemical deterioration and that the emulsions are stable over long periods . In preliminary experiments, sainfoin cells survived 24 hr at -10 degrees C, and shoot-tips survived 48 hr at -10 degrees C . Sainfoin cells, conditioned by growth in medium supplemented with sorbitol, showed enhanced survival after exposure to low temperatures and a lower intracellular freezing point than control cells . Possible reasons for this are discussed. Cell, 1985 Dec, 43(2 Pt 1), 405 - 13 Identification of a specific telomere terminal transferase activity in Tetrahymena extracts; Greider CW et al.; We have found a novel activity in Tetrahymena cell free extracts that adds tandem TTGGGG repeats onto synthetic telomere primers . The single-stranded DNA oligonucleotides (TTGGGG)4 and TGTGTGGGTGTGTGGGTGTGTGGG, consisting of the Tetrahymena and yeast telomeric sequences respectively, each functioned as primers for elongation, while (CCCCAA)4 and two nontelomeric sequence DNA oligomers did not . Efficient synthesis of the TTGGGG repeats depended only on addition of micromolar concentrations of oligomer primer, dGTP, and dTTP to the extract . The activity was sensitive to heat and proteinase K treatment . The repeat addition was independent of both endogenous Tetrahymena DNA and the endogenous alpha-type DNA polymerase; and a greater elongation activity was present during macronuclear development, when a large number of telomeres are formed and replicated, than during vegetative cell growth . We propose that the novel telomere terminal transferase is involved in the addition of telomeric repeats necessary for the replication of chromosome ends in eukaryotes. Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Dec, 48(6), 873 - 92 Cellular and subcellular effects of very heavy ions; Kiefer J; The biological effects of irradiation with ions of masses larger than 40 and energies up to 20 MeV per atomic mass unit are reviewed . The objects are viruses, bacterial spores, yeast and mammalian cells . Experimental parameters include loss of colony forming ability, induction of mutants, chromosomal aberrations, cell cycle progression, inhibition of biochemical activities and the formation of strand breaks . Some of the pertinent physical questions--e.g . track structure--are also discussed . It is shown that with very heavy ions the biological effectiveness is no longer unambiguously related to a single parameter like l.e.t . or Z*2/beta 2 but depends strongly on ion energy . This points to the importance of far-reaching delta-electrons . The analysis indicates also that even with very high l.e.t., cells are not killed by the passage of a single particle through their nucleus . Possible implications of the findings for fundamental radiation biology are outlined. Cell, 1985 Dec, 43(3 Pt 2), 695 - 703 High frequency of homologous recombination in mammalian cells between endogenous and introduced SV40 genomes; Jasin M et al.; We have detected a high frequency of homologous recombination between introduced and chromosomal DNA in mammalian cells . Linear enhancerless SV40 DNA has been transfected into monkey cells that have either one (COS1 cells) or five to seven (COS7 cells) copies of the SV40 early region stably integrated into their genome . Enhancer-containing wild-type SV40 DNA is formed as a result of homologous recombination of the introduced DNA with chromosomal DNA . Up to 25% of the successfully transfected cells produce wild-type virus within 48 hr after transfection . The highest levels of wild-type virus were produced from transfections of molecules that contained a double-strand break at positions of uninterrupted homology with the chromosomal template . This SV40/COS cell system provides a rapid assay for recombination between introduced and genomic DNA. Arch Int Physiol Biochim, 1985 Dec, 93(5), 141 - 50 15N-NMR studies of living systems; Juretschke HP; 15N-NMR spectroscopy has been applied to the study in vivo of nitrogen uptake during primary and secondary metabolism in Streptomyces parvulus . The nitrogen metabolism of Saccharomyces cerevisiae has been studied in a series of experiments in an effort to elucidate the flow of nitrogen along the various competing pathways as well as its dependence on culture conditions . The low NMR sensitivity of the 15N nucleus required, in spite of high isotopic enrichment, quite long acquisition times (10-20 min) . Therefore, an indirect detection method using double quantum 1H-NMR spectroscopy was introduced allowing the selective detection of 15N-bound protons with excellent S/N-ratio in less than a minute. Science, 1985 Nov 29, 230(4729), 1009 - 14 A test of clathrin function in protein secretion and cell growth; Payne GS et al.; Clathrin-coated membranes are intimately associated with a variety of protein transport processes in eukaryotic cells, yet no direct test of clathrin function has been possible . The data presented demonstrate that Saccharomyces cerevisiae does not require clathrin for either cell growth or protein secretion . Antiserum to the yeast clathrin heavy chain has been used to isolate a molecular clone of the heavy chain gene (CHC1) from a library of yeast DNA in lambda gt11 . Clathrin-deficient mutant yeast have been obtained by replacing the single chromosomal CHC1 gene with a disrupted version of the cloned DNA . Cells harboring a nonfunctional chc1 allele produce no immunoreactive heavy chain polypeptide, and vesicles prepared from mutant cells are devoid of clathrin heavy and light chains . Although clathrin-deficient cells grow two to three times more slowly than normal, secretion of invertase occurs at a nearly normal rate . Therefore protein transport through the secretory pathway is not obligately coupled to the formation of clathrin-coated vesicles. Eur J Biochem, 1985 Nov 15, 153(1), 37 - 40 Action of serine carboxypeptidases on endopeptidase substrates, peptide-4-methyl-coumaryl-7-amides; Kunugi S et al.; Carboxypeptidase Y hydrolyzed N-substituted peptide-4-methylcoumarin-7-amides (peptide-NH-Mec) at pH 7 by releasing 7-amino-4-methylcoumarin (NH2-Mec) which was then followed by carboxypeptidase action . In particular, a chymotrypsin-directed substrate, Suc-Leu-Leu-Val-Tyr-NH-Mec, was hydrolyzed by the enzyme with a second-order rate constant of 7200 M-1 s-1, which is compatible with the rate for an anilide substrate and some N-substituted dipeptides . The activity was completely inhibited by phenylmethylsulfonyl fluoride and competitively depressed by the presence of an N-substituted dipeptide . Dependences of kinetic parameters on pH were different from those of carboxypeptidase, esterase, amidase, and anilidase activities . Carboxypeptidases P from Penicillium janthinellum and W from wheat also hydrolyzed some of these peptide-NH-Mec derivatives in a similar manner but at a rather low rate . Thus, the NH2-Mec-releasing activity may be considered to be intrinsic to serine carboxypeptidases in general . Taking into consideration this endopeptidase-like activity of serine carboxypeptidases, fluorogenic substrates should be used carefully to specify endopeptidases in crude extracts. J Theor Biol, 1985 Nov 7, 117(1), 127 - 36 Nucleotide distribution and the recognition of coding regions in DNA sequences: an information theory approach; Almagor H; A method for the recognition of coding-regions along DNA sequences is described . The method is based on the observation, made in several cases, that nucleotide distribution at the third position of the codon is more biased (less random) than that in the other two positions . It is suggested that since nucleotide distribution at the third position is only weakly influenced by the amino acid distribution in the coded protein, there must be some constraints at the DNA level which bias the nucleotide distribution at the third position . The distinction between DNA-level constraints and protein-level constraints is discussed in the frame of Information Theory, and the analysis of the Mitochondrial gene coding for subunit-1 of the yeast cytochrome oxidase is presented. Neurology, 1985 Nov, 35(11), 1582 - 6 Lipid storage myopathy in Kearns-Sayre syndrome; Niebroj-Dobosz I et al.; A 7-year-old girl had external ophthalmoplegia, limb weakness, short stature, hearing loss, pigmentary degeneration of the retina, and increased CSF protein content . Muscle biopsy revealed vacuolar myopathy with accumulation of lipids . Electronmicroscopy showed abnormalities of shape, size, and internal structure of muscle mitochondria . Muscle activity of palmitoyl-CoA synthetase was decreased, and the content of lipids was increased . Serum and muscle carnitine levels were normal, as were muscle carnitine palmitoyltransferase and carnitine acetyltransferase. Nucleic Acids Res, 1985 Oct 25, 13(20), 7195 - 206 5-Methylcytidylic modification of in vitro transcript from the rat identifier sequence; evidence that the transcript forms a tRNA-like structure; Sakamoto K et al.; Previous studies showed that the rat identifier sequence, the rodent type 2 Alu family, the rabbit C family and the bovine or goat 73 bp repeat show remarkable resemblance with a few specific tRNA molecules (Sakamoto and Okada, J . Mol . Evol . in press) . This paper reports 5-methylcytidylic modification of the in vitro transcript from the ID sequence, and provides further evidence that the transcript forms a tRNA-like structure in vitro . Fingerprint analysis and oligonucleotide mapping suggested that the sequence of the oligonucleotide containing 5-methylcytidine is CpCpm5CpUpGp, which corresponds to the extra and T psi stem regions in the secondary structure of phenylalanine tRNA . Since the sequence of the corresponding oligonucleotide in the phenylalanine tRNA is Cpm5CpCpUpGp, these results suggests that a tRNA (cytosine-5) methyl transferase recognizes the secondary structure of the transcript from the ID sequence rather than the primary sequence . The significance of this modification in relation to the functional role of the ID sequence is discussed. J Mol Biol, 1985 Oct 20, 185(4), 659 - 80 Excision-amplification of mitochondrial DNA during senescence in Podospora anserina . DNA sequence analysis of three unique "plasmids"; Cummings DJ et al.; During senescence in the filamentous fungus Podospora anserina, specific regions of the mitochondrial genome, termed senDNA are excised, ligated and amplified . We have cloned in their entirety three such autonomously replicating plasmids, alpha, beta and epsilon senDNA . None of these plasmids displayed cross-hybridization nor did we detect any significant DNA homology by computer analysis . The complete DNA sequence of the 2.5 kb alpha, the 5.5 kb epsilon and about 3.4 kb of the 9.8 kb beta senDNA is presented (kb = 10(3) base-pairs) . These sequences were analyzed for the presence of consensus sequences common to introns, and it was found that alpha senDNA has the characteristics of a group II intron, epsilon senDNA contains three group I introns, and beta senDNA did not show relevant sequences in the 3.4 kb examined . Comparison of the 5' and 3'-flanking sequences of alpha senDNA with oxi 3 (Co I) amino acid sequences from Neurospora crassa and Saccharomyces cerevisiae revealed significant homology and provided strong support that the excised alpha senDNA itself consists entirely of an intron . Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected . beta senDNA contained four tRNA sequences, aspartic acid, serine, valine and tryptophan, and sequences homologous to URFC (untranslated reading frame C) as well as two new URFs . epsilon senDNA contained sequences homologous to ATPase 8 and URFl; URFl was interrupted by three group I introns . The excision site sequences, as located by S1 nuclease mapping were unique for each senDNA . Analysis for repeated units showed that each plasmid contained elements which could be involved in secondary structure required for the alignment of distal ends preparatory to excision . These results are interpreted in terms of the structural requirements of mobile elements including the possible involvement of reverse transcriptase in the excision-ligation-amplification process. J Biol Chem, 1985 Oct 5, 260(22), 12320 - 7 Determination of DNA sequences essential for FLP-mediated recombination by a novel method; Gronostajski RM et al.; The yeast 2-micron circle plasmid encodes a protein, FLP, that mediates site-specific recombination across the two FLP-binding sites of the plasmid . We have used a novel technique, "exonuclease-treated substrate analysis," to determine the minimal duplex DNA sequence needed for this recombination event . A linear DNA containing two FLP sites in a direct orientation was treated with the double-strand specific 3'-exonuclease, exonuclease III, to generate molecules with a nested set of single-strand deletions that extended into one of the FLP sites . The DNA was then end-labeled at the sites of the deletions and used as a substrate for recombination in vitro . FLP-mediated recombination between two FLP sites excised a restriction endonuclease cleavage site from the DNA . Comparison of the fragments produced by restriction enzyme digestion of untreated and FLP-treated DNA showed to the nucleotide the duplex DNA sequence required for FLP-mediated recombination . To examine essential sequences in the opposite DNA strand, similar experiments were done using the 5'-exonuclease encoded by phage T7 . The minimal essential duplex DNA sequence lies within the region of the FLP site that was previously shown to be protected from nuclease digestion in the presence of FLP . A modified form of this technique can be used to study the minimal sequence requirements of site-specific DNA binding proteins. Biochem J, 1985 Oct 1, 231(1), 165 - 9 Irreversible inhibition of putrescine-stimulated S-adenosyl-L-methionine decarboxylase by berenil and pentamidine; Karvonen E et al.; The putrescine-stimulated S-adenosyl-L-methionine decarboxylases from rat liver and yeast were strongly inhibited by Berenil and to a lesser extent by Pentamidine . Ten times greater drug concentrations were needed to achieve a similar level of inhibition of a Mg2+-stimulated bacterial enzyme . The inhibition was irreversible in that extensive dialyses or precipitation with (NH4)2SO4 did not restore enzyme activity . Putrescine did not protect the enzyme against Berenil, but adenosylmethionine either alone or with putrescine partially protected the irreversible action of Berenil . The compound 4,4'-diamidinodiphenylamine, which differs from Berenil only in lacking the azo group between benzene rings, was a weaker inhibitor than Berenil, and its inhibition was reversible . Berenil also inhibited the activity of adenosylmethionine decarboxylase in vivo, by depressing the activity of the enzyme in normal rat liver, for at least 24 h after a single injection (50 mg/kg body wt.) of the drug. Biosci Rep, 1985 Oct-Nov, 5(10-11), 847 - 54 Studies on N alpha-acylated proteins: the N-terminal sequences of two muscle enolases; Chin CC et al.; A standard procedure for the identification of the N-terminal amino acid in N alpha-acylated proteins has been developed . After exhaustive proteolysis, the amino acids with blocked alpha-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I . Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid . A survey of acylamino acid substrates showed that acylase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl-and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val . Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, N alpha-acyl-derivatives of Arg, Lys or His . Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected . When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle . The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins . When such peptides were treated with 1 N HCl at 110 degrees for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing . The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase. Biochem Int, 1985 Oct, 11(4), 617 - 25 Action of Pennisetum typhoides double-stranded ribonuclease on viral ds RNAs; Maran A et al.; A ribonuclease that preferentially cleaves natural and synthetic double-stranded (ds) RNAs has been partially purified from Pennisetum typhoides . The enzyme degrades {3H}poly(rA) X poly(rU) to acid-soluble products . The ds RNase does not degrade RNA:DNA hybrid but appears to have about 18% activity against single-stranded (ss) RNAs under the assay conditions used for the cleavage of ds RNAs . The RNase has a molecular weight of 35,000 daltons as determined from gel filtration using Sephadex G-200 . The ds RNase shows an absolute requirement for divalent cations Mg++ or Mn++ and monovalent cations K+ or Na+ . The specificity of the enzyme towards ds RNA template is supported by the inhibition of cleavage of ds RNAs by ethidium bromide and Penicillium chrysogenum viral ds RNA . The enzyme preparation acts on ds RNAs isolated from Saccharomyces cerevisiae and P . chrysogenum virus . The purified ds RNase also cleaves the in vitro transcriptional products of adenovirus DNA and this activity is inhibited by 5 mM ethidium bromide suggesting that ds regions of adenovirus RNA are involved in the cleavage process. Mutat Res, 1985 Oct, 147(5), 231 - 5 Study of the optimal temperature for the liver microsomal assay with mice S9 fractions; Mannironi C et al.; The effect of temperature on enzymatic activity and stability was studied with respect to the monooxygenase activities of aminopyrine-N-demethylase (APD) and p-nitroanisole O-demethylase (pNAD) under incubation conditions for the liver microsomal assay . The activities of S9 liver fractions of mice induced with sodium phenobarbital and beta-naphthoflavone were determined during a period of preincubation in a range of temperatures from 30 to 44 degrees C . The greatest value of the mean specific activity was found at 40-42 degrees C for both APD and pNAD . The rapid increase of lipid peroxidation after 1 h of incubation at temperatures higher than 42 degrees C can provide an explanation of the enhancement of the rate of inactivation . In order to determine whether biological response is affected by the modifications induced by temperature in the metabolic activating system, tester strain D7 of Saccharomyces cerevisiae was used to assay the genetic activity of the well known premutagenic agent cyclophosphamide by incubating the mixtures both at the traditional temperature of 37 degrees C and at 42 degrees C . We suggest that the use of more favourable conditions for LMA with respect to enzymatic activity, than the traditional ones could improve the reliability and the sensitivity of such tests. Muscle Nerve, 1985 Oct, 8(8), 672 - 5 Beta-oxidation enzymes in normal human muscle and in muscle from a patient with an unusual form of myopathic carnitine deficiency; Trevisan CP et al.; In a reported patient with myopathic carnitine deficiency, addition of exogenous carnitine to muscle homogenates failed to correct palmitate oxidation, and oral carnitine was of no clinical benefit . In a muscle biopsy from this patient, we found that, in contrast to the marked deficiency of free carnitine (3% of normal) short- and medium-chain acylcarnitines were in the normal range and long-chain acylcarnitine was increased almost four times . As this result confirmed the hypothesis of a muscle defect of mitochondrial oxidation of palmitate, all eight enzymes of beta-oxidation were measured spectrophotometrically in the muscle extract . None of them was found to be defective . These data suggest that the underlying biochemical abnormality in this patient may be a deficiency of the carnitine-acylcarnitine translocase system or a defective interaction between acyl-CoA dehydrogenase and its flavoprotein coenzyme. Cryobiology, 1985 Oct, 22(5), 446 - 56 Freeze denaturation of enzymes and its prevention with additives; Tamiya T et al.; Freeze inactivation of LDH, MDH, ADH, G-6-PDH, and PK and its prevention with additives such as sodium glutamate and albumin were studied . LDH, MDH, ADH, G-6-PDH, and PK, each lost their activity during frozen storage at -20 degrees C . The speed of the inactivation differed in each . The stability of the enzymes increased with the increase of the enzyme concentration . Sodium glutamate and albumin prevented the freeze inactivation . While the activity of the LDH solution frozen without additives was almost lost during a day of frozen storage, those frozen with either glutamate (0.2 M) or albumin (0.1%) added decreased less quickly . The residual activity after 1 day was 50% the initial prefreeze value for the former and 10% for the latter, respectively . Combined use of glutamate and albumin prevented the inactivation the best and maintained the initial activity almost completely over 6 weeks . The enzymes tested lost some part of their activity when their solutions were diluted by the media . This inactivation was prevented to a significant extent by the addition of sodium glutamate and/or albumin to the diluting media. J Biomol Struct Dyn, 1985 Oct, 3(2), 409 - 21 Probing stability and dynamics of proteins by protease digestion . I: Comparison of protease susceptibility and thermal stability of cytochromes c; Endo S et al.; Protease susceptibility of homologous proteins in their native conformations was studied . This work aims to establish a broad and quantitative basis for the utilization of protease digestion to analyze the local stability of native proteins . Using high-performance liquid chromatography (HPLC) the time course of the proteolytic degradation of intact proteins was quantitatively traced . Rapid separation of peptide fragments with HPLC made possible the elucidation of sequential digestion originating from the cleavage at a very few sites which are locally unstable in the protein structure . Using four serine proteases, chymotrypsin, trypsin, elastase and subtilisin BPN', we found some common trends in proteolysis for a group of proteins of the cytochrome c family . By comparing of the proteolysis and thermal denaturation with ten homologous cytochromes c extracted from horse, beef, Candida krusei, Saccharomyces cerevisiae, chicken, tuna, pigeon, rabbit, dog and rat, protease susceptibility was related to locally unfolding states intrinsic to the native conformation. Biochemistry, 1985 Sep 24, 24(20), 5328 - 33 Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of 3-phosphoglycerate kinase; Moore JM et al.; The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy . Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme . The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor . The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II) . EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion . EPR data for complexes with the two epimers of {alpha-17O}ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site . EPR spectra for Mn(II) in the enzymic complex with (Rp)-{alpha-17O}ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-{alpha-17O}ADP complexes are indistinguishable from those for matched samples with unlabeled ADP . These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP . Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3-phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Biochem, 1985 Sep 16, 151(3), 497 - 504 Plant peroxidases . Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase; Welinder KG; The amino acid sequences of the 51% different horseradish peroxidase HRP C and turnip peroxidase TP 7 have previously been completed by us, but the three-dimensional structures are unknown . Recently the amino acid sequence and the crystal structure of yeast cytochrome c peroxidase have appeared . The three known apoperoxidases consist of 300 +/- 8 amino acid residues . The sequences have now been aligned and show 18% and 16% identity only, between the yeast peroxidase and plant peroxidase HRP C and TP 7, respectively . We show that different structural tests all support similar protein folds in plant peroxidases and yeast peroxidase and, therefore, a common evolutionary origin . The following tests support this thesis: (a) predicted helices in the plant peroxidases follow the complex pattern observed in the crystal structure of cytochrome c peroxidase; (b) their hydropathic profiles are similar and agree with observed buried and exposed peptide chain in cytochrome c peroxidase; (c) half-cystines which are distant in the amino acid sequence of plant peroxidases become spatial neighbours when fitted into the cytochrome c peroxidase model; (d) the two-domain structure proposed from limited proteolysis of apoperoxidase HRP C is observed in the crystal structure of cytochrome c peroxidase . The similarities and differences of the plant and yeast peroxidases and the reactive side chains of a plant peroxidase active site are described . The characteristics of Ca2+-binding sequences, derived from several superfamilies, are applied to predict the Ca2+-binding sequences in plant peroxidases. Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 949 - 55 The adjuvant effects of mycoviral dsRNA and polyinosinic:polycytidylic acid on the murine immune response; Wright CL et al.; By comparing, under the same experimental conditions, the effects of naturally occurring mycoviral dsRNA with those of the synthetic dsRNA, polyinosinic:polycytidylic acid, we were able to determine if the source of the dsRNA would modify its immunomodulating properties . Mycoviral dsRNA, but not the synthetic dsRNA, significantly enhanced the hemagglutinating antibody response to sRBC in C57B1/6 mice . Although both dsRNA preparations significantly increased the rate of rejection of heterologous skin grafts by recipient mice when compared to controls, mycoviral dsRNA induced higher interferon titers than the synthetic dsRNA . This study showed that mycoviral dsRNA was a more potent adjuvant than polyinosinic:polycytidylic acid for both humoral and cellular immune responses. Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 883 - 91 Fast and efficient method for detection and estimation of proteins; Kumar BV et al.; A quick, simple, inexpensive and sensitive method is described to stain and quantitate proteins on nitrocellulose papers . The proteins may be spotted or transferred from polyacrylamide gels by Western blotting . The procedure involves non-radioactive iodination of the polypeptides by chloramine T and potassium iodide followed by detection of bound iodine with starch . The method is more sensitive and much quicker than Coomassie brilliant blue staining and may be used for quantitation or detection of proteins in unknown samples . Another major advantage of this procedure is that ionic or nonionic detergents, although at higher concentrations causing the sample to disperse more broadly in the membranes, do not affect the staining procedure . Further, this method may be used for detection of proteins bound to papers that have high affinity for proteins such as the Zeta probe membranes. Nucleic Acids Res, 1985 Sep 11, 13(17), 6035 - 47 Cloning of the chick hsp 90 cDNA in expression vector; Catelli MG et al.; A cDNA clone for the 90kDa heat-shock protein, which we have recently identified as a component of steroid hormone receptors in their heteromeric 8S form, was isolated by direct immunological screening of a chicken smooth muscle cDNA expression library, prepared in the expression plasmids pUC8 and pUC9 . Using polyclonal and monoclonal antibodies against the 90kDa protein a colony was identified that reacted with both antibodies . Plasmid 9.11 (p9.11, approximately 1100 base pair insert) was found to hybrid-select mRNA for the 90kDa heat-shock protein . Northern blot analysis revealed that RNA isolated from various chicken tissues contain a single transcript of approximately 3 Kb hybridizing to a {32P}labelled cDNA probe made from p9.11 . Heat-shock treatment of chick embryonic fibroblasts resulted in increased steady-state levels of a 3 Kb transcript in both poly A+ and poly A- RNA fractions . Southern blot analysis of chicken genomic DNA indicated that the cDNA hybridizes to a single copy sequence . Sequence data show that the p9.11 cDNA displays a high degree of homology with the 5' portion of yeast heat shock protein 90 cDNA. Biochim Biophys Acta, 1985 Sep 11, 836(2), 176 - 84 Purification and characterization of a phosphatidylinositol transfer protein from human platelets; George PY et al.; We report the purification of a phospholipid transfer protein from human platelets . This protein preferentially transfers phosphatidylinositol, with phosphatidylcholine and phosphatidylglycerol being transferred to a lesser extent . Phosphatidylethanolamine is not transferred . Transfer activity is detected by measuring the transfer of radiolabeled phospholipids between two populations of small unilamellar vesicles . The protein was purified approximately 1000-fold over the platelet cytosol by chromatography on Sephadex G-75, sulfooxyethyl cellulose, and hydroxylapatite . The molecular weight of this protein appears to be 28 000 as determined by gel filtration chromatography . When the purified protein is analyzed on sodium dodecyl sulfate-polyacrylamide gels, two major components and several minor ones are observed . The molecular weight of the two major bands are 28 600 and 29 200 . Isoelectric focusing of the platelet cytosol yielded phosphatidylinositol and phosphatidylcholine transfer activity at pH 5.6 and 5.9 . The platelet phospholipid transfer protein is able to catalyze the transfer of phosphatidylinositol and phosphatidylcholine between vesicles and human platelet plasma membranes . One possible physiological role for this transfer protein is an involvement in the rapid turnover of inositol-containing lipids which occurs upon exposure of platelets to various stimuli. J Androl, 1985 Sep-Oct, 6(5), 265 - 70 Activation of palmitic acid by human spermatozoa; Jones RE et al.; Human spermatozoa were studied to determine if a long chain fatty acid, CoASH ligase (AMP) (E.C . 6.2.1.3), was present . Ligase activity was measured with a radioligand millipore filter technique and was readily detectable in spermatozoa or in the protein fraction extracted with Triton X-100, but was not present in seminal plasma . The assay was optimized for pH, protein concentration, and incubation time . Activity was dependent upon palmitic acid, ATP, coenzyme A, and a divalent cation . Sperm ligase appeared similar to the ligase characterized from other tissues by sharing a common pH optimum (approximately 8.0-8.4), and a preference for magnesium over manganese in the incubation media. Anal Biochem, 1985 Sep, 149(2), 507 - 15 An affinity column for the purification of prenyltransferases; Bartlett DL et al.; Farnesyl pyrophosphate synthetase (EC 2.5.1.1) from chicken liver, pig liver, and yeast has been purified to homogeneity in a single chromatographic step by affinity chromatography . The affinity ligand, geranylmethylphosphonophosphate, is linked to Affi-Gel 10 through the phosphonophosphate moiety . The affinity gel is stable chemically and the internal phosphonophosphate linkage is not hydrolyzed by nonspecific phosphatases . A single column has been used repeatedly for over a year with no degradation in its performance . A typical purification only requires 2 days and gives a 500- to 600-fold purification of enzyme from a crude ammonium sulfate precipitate. Int J Biomed Comput, 1985 Sep, 17(2), 107 - 14 Spatial distribution of cell fluorescence determined with a diode-matrix (32 x 32 elements) and computer controlled epifluorescence microscopy; Sundqvist T et al.; A photodiode-matrix with 32 x 32 light-sensitive elements has been used to determine spatial fluorescence intensity of Rhodamine-labelled yeast cells (Saccharomyces cerevisiae) . The matrix with electronics was built into one of the oculars of an epifluorescence microscope, and the fluorescence intensity scanned at intervals, determined from a desk-computer . The signals from one image were amplified and stored in a buffer-memory before being put into an auxiliary memory . The sensitivity of each element was calibrated, and linear regression used to determine true fluorescence intensity (256 levels) . After background subtraction the spatial intensity was presented on a plotter either on a 0-9 scale, or on a three-dimensional plot (x-y-intensity) . The effect of continuous UV-microphotolysis on the fluorescence of Rhodamine-labelled yeast was also determined. Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6250 - 4 Cloning, sequence, and expression of a human granulocyte/macrophage colony-stimulating factor; Cantrell MA et al.; Human granulocyte/macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is essential for the in vitro proliferation and differentiation of precursor cells into mature granulocytes and macrophages . In this report we have used a mouse GM-CSF cDNA clone to isolate human GM-CSF clones from libraries made from HUT-102 messenger RNA and mitogen-stimulated T-lymphocyte messenger RNA . The human cDNA clones contained a single open-reading frame encoding a protein of 144 amino acids with a predicted molecular mass of 16,293 daltons and showed 69% nucleotide homology and 54% amino acid homology to mouse GM-CSF . One of these cDNA clones was shown to direct the synthesis of biologically active GM-CSF using a yeast expression system . The gene for human GM-CSF appears to exist as a single-copy gene. Proc Natl Acad Sci U S A, 1985 Sep, 82(17), 5875 - 9 Two-micrometer circle site-specific recombination: the minimal substrate and the possible role of flanking sequences; Jayaram M; The 2-mum circle DNA of yeast encodes a site-specific recombination system (FLP recombination) . The recombination region had been mapped earlier to a 65-base-pair (bp) segment within the 599-bp-long inverted repeats of the molecule . I have shown that the "minimal" FLP substrate resides in a 13-bp dyad symmetry plus an 8-bp core located within the 65-bp recombination region . Further, as determined by different in vivo assays, sequences extraneous to the minimal FLP site and the 65-bp recombination region can affect the efficiency of the recombination reaction. J Bacteriol, 1985 Sep, 163(3), 1180 - 5 Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization; Roncero C et al.; Calcofluor White (a fluorochrome dye) affected the growth of Geotrichum lactis by causing lysis of cells at the hyphal tips . This effect was prevented by the presence of an osmotic stabilizer . The growth of Saccharomyces cerevisiae was also affected, and multicellular aggregates were formed because of incomplete separation of mother and daughter cells; fluorescence microscopy indicated the presence of abnormally thick septa . The formation of rudimentary wall material by G . lactis protoplasts was promoted by Calcofluor or Congo red (another dye) . The rate of chitin synthesis in protoplasts and growing cells was enhanced by both dyes . In contrast, both dyes inhibited chitin and beta (1,3)-glucan synthases in isolated cell-free systems . Degradation rates for beta (1,3)-glucan or chitin were not affected significantly by the dyes . Wheat germ agglutinin also affected chitin synthesis in protoplasts. Cell, 1985 Sep, 42(2), 507 - 17 Ty element transposition: reverse transcriptase and virus-like particles; Garfinkel DJ et al.; We have found reverse transcriptase activity and virus-like particles only in yeast cells that contain a galactose-promoted Ty element induced on galactose . The cofractionation of reverse transcriptase, genomic-length Ty RNA, and a Ty-specified protein antigen in a particulate fraction and the ability of this complex to synthesize specifically a product that is homologous to the entire Ty suggest that reverse transcription of Ty RNA takes place in the particle . The absence of appreciable levels of reverse transcriptase and particles in uninduced cells despite the presence of at least 35 copies of chromosomal Ty elements suggest that some of these elements may be defective . The numerous virus-like particles visible in thin sections of Ty transposition-induced cells appear not to be infectious . These particles resemble the intracisternal A-type particles of the mouse and copia particles of Drosophila . The results support the idea that Ty elements and retroviruses share a common origin. Chem Phys Lipids, 1985 Aug 30, 38(1-2), 3 - 16 Phosphatidylinositol transfer proteins: structure, catalytic activity, and physiological function; Helmkamp GM Jr; Among the diverse lipid transfer proteins which are found in tissues and biological fluids are those which exhibit a specificity toward phosphatidylinositol and phosphatidylcholine, with a preference for the former . Phosphatidylinositol transfer proteins (PI-TPs) have been purified from several eukaryotic sources; those present in bovine brain and heart have been extensively studied . This review examines the tissue distribution of PI-TPs and the means by which transfer activity is measured using natural and artificial membranes . The interaction of these proteins with lipid monolayers and bilayers is discussed in terms of phospholipid fatty acyl and polar head group compositions . The inhibition of transfer activity by sulfhydryl agents and amphiphilic amines is summarized . The metabolism of the phosphoinositides is considered and a role for PI-TPs is proposed. Biochim Biophys Acta, 1985 Aug 22, 836(1), 80 - 8 Lignoceroyl-coenzyme A synthetase from developing rat brain: partial purification, characterization and comparison with palmitoyl-coenzyme A synthetase activity and liver enzyme; Nagamatsu K et al.; As part of a long-term study of sphingolipid metabolism in brain, we have purified and partially characterized a long-chain acyl-CoA synthetase from microsomes of developing rat brain and compared it with the hepatic microsomal enzyme from the same animals . Both enzymes were solubilized from microsomes by treatment with Triton X-100 and then chromatographed successively on Blue-Sepharose and DEAE-Sepharose . Blue-Sepharose chromatography yielded a single peak with acyl-CoA synthetase activity, whereas DEAE-Sepharose chromatography of both brain and liver preparations yielded two peaks . Elution patterns of lignoceroyl-CoA synthetase and palmitoyl-CoA synthetase activities were identical throughout these steps and were similar in brain and liver . Gel filtration of each DEAE-Sepharose fraction on Sephadex G-200 also yielded two peaks of activity . The more rapidly eluted material contained much more lignoceroyl-CoA synthetase activity, while the activity for palmitoyl-CoA synthetase was higher in slower eluting peaks . In all preparations the ratio of lignoceroyl-CoA synthetase activity to palmitoyl-CoA synthetase activity was much higher in brain than in liver . These results suggest that although the brain acyl-CoA synthetase is chromatographically similar to the liver enzyme, there are differences in substrate specificity. Arch Biochem Biophys, 1985 Aug 15, 241(1), 243 - 51 Structural analyses of various Cu2+, Zn2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy; Lepock JR et al.; The thermal denaturation profile of the Cu2+, Zn2+ metalloenzyme, bovine superoxide dismutase, consists of two primary components, the major component denatures irreversibly at Tm = 104 degrees C with a total enthalpy (delta Hcal) of 7.30 cal/g . Reduction of Cu(II) to Cu(I) with potassium ferrocyanide lowers Tm to 96 degrees C and delta Hcal to 6.96 cal/g . The apo-form of bovine superoxide dismutase (both Cu and Zn removed) denatures at 60 degrees C with an enthalpy only one-half that of the holo-form . The reduced thermal stability, which indicates a greater ability to change conformation, may explain the previously observed much greater membrane binding of the apo-enzyme . Reconstitution with Zn2+, Cu2+, or Zn2+ and Cu2+ raises Tm to 80, 89, or 102 degrees C, respectively, with corresponding increases in the enthalpy . Thus, the metal ions considerably stabilize the enzyme and must somewhat affect conformation . The effect of Cu2+ alone is greater than that of Zn2+, although both are needed for full stability . Raman spectroscopy indicates little difference in secondary structure between the apo- and holo-forms, implying that the increased stability due to metal binding is not caused by an extreme structural reorganization . The value of Tm of canine and yeast superoxide dismutase is also lowered by reduction of Cu(II) . The reduced form of the yeast enzyme denatures irreversibly, as do all forms of the bovine and canine enzymes, but the oxidized form is unique in that it denatures reversibly . Thus, the copper ion must be oxidized for renaturation and appears to act as a nucleation site. Nature, 1985 Aug 15-21, 316(6029), 641 - 3 Mitochondrial class II introns encode proteins related to the reverse transcriptases of retroviruses; Michel F et al.; Organelle introns share several distinctive features that set them apart from their counterparts in nuclear-encoded pre-messenger RNAs (reviewed in ref . 1): their termini do not obey the GU...AG rule; the introns are 'structured' (members of the same family or 'class' can theoretically adopt very similar RNA secondary conformations and several of the postulated pairings have been confirmed by studies of splicing mutants and their revertants (see, for example, ref . 4); many introns from both classes contain long open reading frames . We report here that the proteins potentially encoded by four class II introns are related to several RNA-dependent polymerases of viral and transposable element origins. FEBS Lett, 1985 Aug 5, 187(2), 349 - 53 What family of ATPases does the vacuolar H+-ATPase belong to? Lichko LP, Okorokov LA. Polyacrylamide gel electrophoresis (PAGE) of partially purified ATPase from vacuoles of Saccharomyces carlsbergensis under non-dissociating conditions revealed 3 bands with ATPase activity . Further PAGE in dissociating conditions showed the similarity in composition of these 3 ATPase preparations . They were assumed to contain the same vacuolar ATPase exhibiting, however, different electrophoretic mobility due to the formation of enzyme complexes with different proteins and phospholipids . The ATPase preparation with the highest electrophoretic mobility contained 6 subunits of 75, 62, 16, 14, 12 and 9 kDa . Inhibitors of vacuolar ATPase {14C}DCCD and {14C}NEM bound to a 9 kDa polypeptide, while {14C}DES associated with a polypeptide of 75 kDa . A partially purified preparation of the vacuolar ATPase was not phosphorylated by {gamma-32P}ATP under conditions when plasma membrane ATPase formed a phosphorylated intermediate . Our results show that vacuolar H+-ATPase consists of several polypeptides, does not form the phosphorylated intermediate, and evidently represents a new type of H+-ATPase of yeast. J Bioenerg Biomembr, 1985 Aug, 17(4), 251 - 61 Further studies on the binding of DCCD to cytochrome B and subunit VIII of complex III isolated from beef heart mitochondria; Beattie DS et al.; Complex III (the cytochrome b-c1 complex) from beef heart mitochondria was incubated with {14C}DCCD for various periods of time . The polypeptide profile of the complex was compared in both stained gels and their autoradiograms when three different methods were used to terminate the reaction . Precipitation with ammonium sulfate resulted in the formation of a new band with an apparent molecular weight of 39,000 in both incubated samples and the zero time controls . Reisolation of the complex by centrifugation through 10% sucrose or by precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunit peptides of the complex . Subunit III (cytochrome b) and subunit VIII were the only bands labeled after termination of the reaction by centrifugation through sucrose, while both ammonium sulfate and trichloroacetic precipitation resulted in nonspecific labeling of several other subunits of the complex and increased labeling of subunit VIII relative to subunit III . Preincubation of the complex with antimycin prior to treatment with {14C}DCCD resulted in a 50% decrease in the binding of DCCD to both cytochrome b and subunit VIII . Furthermore, treatment of the complex III with DCCD resulted in a change in the red shift observed after antimycin or myxothiazol addition to the dithionite-reduced complex resulting in a broad peak with no sharp maximum . These results provide further confirmation that DCCD binds preferentially to cytochrome b and subunit VIII of complex III from beef heart mitochondria and suggest that cytochrome b may play a role in proton translocation. Biochem Med, 1985 Aug, 34(1), 60 - 9 Human spleen dihydroorotate dehydrogenase: a study of inhibition of the enzyme; Gero AM et al.; Seventy-one pyrimidine analogs have been tested as possible inhibitors of human spleen mitochondrial dihydroorotate dehydrogenase . Of these nine were demonstrated to be effective inhibitors of the enzymic activity . Two compounds, dihydro-5-azaorotate and 6-thiobarbiturate appeared to be specific inhibitors of the DHO-DHase . In addition, three compounds, 5-azaorotate, 5-bromoorotate, and barbiturate were also inhibitory against the two subsequent enzymes of the pathway, orotate phosphoribosyltransferase and orotidylate decarboxylase, so that they could act against three enzymes of the mammalian pyrimidine de novo biosynthetic pathway. Science, 1985 Jul 5, 229(4708), 52 - 4 Dominance in physiological phenotypes and fitness at an enzyme locus; Hilbish TJ et al.; Aminopeptidase-I allozymes, which are products of the Lap locus in the marine mussel, Mytilus edulis, differ in their catalytic efficiencies . These biochemical differences result in genotype-specific rates of change in the free amino acid pool, that is, cell volume regulation, when mussels are subjected to changes in salinity . A high degree of dominance was found among genotypes for these biochemical and physiological phenotypes . Selection models that incorporate dominance adequately predict observed genotypic properties at the Lap locus among natural populations that exhibit clinical allele frequency . This suggests that a high degree of dominance for fitness must also occur at this locus in natural populations . These results provide additional evidence that the maintenance of an allele frequency cline is operating by natural selection at the Lap locus. Biochemistry, 1985 Jul 2, 24(14), 3459 - 64 Refolding a disulfide dimer of cytochrome c; Bryant C et al.; A covalent dimer of Saccharomyces cerevisiae iso-1 cytochrome c is stabilized by an interchain disulfide bond involving the cysteine residue penultimate to the C-terminus . The individual chains in the dimer appear to retain the tertiary structural features characteristic for monomeric cytochrome c albeit with some perturbation . The dimer is reversibly denatured by heat, urea, or guanidine hydrochloride in a single cooperative transition whose midpoint is less than that of the monomeric protein . The kinetic profile observed for the refolding of the denatured dimer is characteristic for monomeric cytochromes except for a markedly enhanced slow-phase amplitude. Biochemistry, 1985 Jul 2, 24(14), 3521 - 5 Fatty acids bound to unilamellar lipid vesicles as substrates for microsomal acyl-CoA ligase; Noy N et al.; Palmitate incorporated into single-layered vesicles of phosphatidylcholine was used as a substrate for palmitoyl coenzyme A ligase (palmitoyl-CoA ligase) in microsomes from rat liver . This was done in order to avoid the use of detergents for dispersal of the water-insoluble palmitate and the possibility of precipitating palmitate added to the aqueous assay as a salt suspension . The activity of the ligase measured when palmitate was added to assays as a component of phospholipid vesicles was 10-40-fold greater vs . activities reported in the literature using other methods for adding fatty acids to the assay system . Phospholipids, however, had no direct effect on the activity of palmitoyl-CoA ligase . The data indicate, therefore, that the activity of this enzyme has been underestimated because of the manner in which fatty acid was added to the assay, which has a significant effect on the activity of the ligase . It is shown too that the rate of spontaneous transfer of palmitate from unilamellar vesicles of phosphatidylcholine to microsomes via a hydrated intermediate is far more rapid than the inherent catalytic activity of the fatty acyl-CoA ligase . The data also suggest that the membrane-associated pool of fatty acid and not fatty acid in the aqueous phase of the assay is the pool of substrate interacting with the ligase. Mutat Res, 1985 Jul, 154(1), 29 - 48 The genotoxicity of selenium; Shamberger RJ; Selenium at nutritional levels has been shown to have numerous anticarcinogenic or preventative effects against carcinogen-induced breast, colon, liver and skin cancer in animals . Many of these anticarcinogenic effects have been summarized . In addition, numerous mutagenic and antimutagenic effects of selenium compounds have been reported . Some of the selenium compounds frequently tested for mutagenicity are listed in Table 1 . Because of the numerous reported anticarcinogenic and preventative effects of selenium, many individuals are supplementing their diets with amounts of selenium that are greater than the recommended daily requirement . Selenium is also used widely in industrial products such as selenium rectifiers, photoelectric batteries, alloys and paints . Because selenium at higher levels is known to be toxic, there should be a greater understanding about its genotoxic as well as its beneficial effect . The object of this review is to summarize experimental evidence both for the antimutagenic and the mutagenic effect of selenium. Mol Cell Biol, 1985 Jul, 5(7), 1714 - 21 Direct and indirect gene replacements in Aspergillus nidulans; Miller BL et al.; We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation . A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+ . Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains . An A . nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+ . Approximately 30% of the transformants were simultaneously argB- . The null argB allele had replaced the wild-type allele in a majority of these strains . The A . nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid . A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected . All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy . Thus, it is possible with A . nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae. Mol Cell Biol, 1985 Jul, 5(7), 1630 - 8 Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins; Mount SM et al.; We have determined the complete nucleotide sequence of the copia element present at the white-apricot allele of the white locus in Drosophila melanogaster . This transposable element is 5,146 nucleotides long and contains a single long open reading frame of 4,227 nucleotides . Analysis of the coding potential of the large open reading frame, which appears to encode a polyprotein, revealed weak homology to a number of retroviral proteins, including a protease, nucleic acid-binding protein, and reverse transcriptase . Better homology existed between another part of the copia open reading frame and a region of the retroviral pol gene recently shown to be distinct from reverse transcriptase and required for the integration of circular DNA forms of the retroviral genome to form proviruses . Comparison of the copia sequence with those of the Saccharomyces cerevisiae transposable element Ty, several vertebrate retroviruses, and the D . melanogaster copia-like element 17.6 showed that Ty was most similar to copia, sharing amino acid sequence homology and organizational features not found in the other genetic elements. Biochim Biophys Acta, 1985 Jun 11, 816(1), 93 - 101 Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid; Dugan JM et al.; The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins . This pathway has been characterized further by studying the activation and incorporation of {9,10(n)-3H}oleic acid and transesterification of {1-14C}oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts . Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars . Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid . Compared to ghosts, the ability of inside-out vesicles to activate and incorporate {9,10(n)-3H}oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify {1-14C}oleoyl-CoA to phospholipid is diminished by over 50% . These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities . This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation. J Biol Chem, 1985 Jun 10, 260(11), 6836 - 42 Interaction of inorganic vanadate with glucose-6-phosphate dehydrogenase . Nonenzymic formation of glucose 6-vanadate; Nour-Eldeen AF et al.; Inorganic vanadate (Vi) activates catalysis by glucose-6-phosphate dehydrogenase of the oxidation of glucose by NADP+ . As the concentration of Glu-6-P dehydrogenase is increased, the rate of the vanadate-activated glucose oxidation becomes less sensitive to increases in enzyme concentration . The rate of glucose oxidation in the absence of Vi increases linearly with Glu-6-P dehydrogenase concentration . These results are interpreted in terms of nonenzymic formation of glucose 6-vanadate . At high enzyme concentration, vanadate ester formation becomes partially rate-limiting, and extrapolation to infinite Glu-6-P dehydrogenase concentration allows determination of the second order rate constant for formation of the ester . In separate experiments designed to test the proposed mechanism, it was found that Vi, at concentrations at which it strongly activates catalysis by Glu-6-P dehydrogenase of glucose oxidation, has no effect on the rates of oxidation of glucose 6-phosphate or 6-deoxyglucose catalyzed by Glu-6-P dehydrogenase . Sulfate, which is known to activate glucose oxidation and to inhibit glucose 6-phosphate oxidation, strongly activates 6-deoxyglucose oxidation . These experiments show that the 6-hydroxyl group of glucose is essential for the observed activation by Vi and are also consistent with the formation of glucose 6-vanadate . Also, the rate of the sulfate-activated glucose oxidation increases linearly with Glu-6-P dehydrogenase concentration . These results are consistent with the proposed mechanism for sulfate activation which involves sulfate binding to the enzyme (Anderson, W . B., Horne, R . N., and Nordlie, R . C . (1968) Biochemistry 7, 3997-4004) . The second order rate constant calculated for formation of glucose 6-vanadate at pH 7.0 is 2.4 M-1 s-1 . The corresponding values for glucose 6-phosphate and glucose 6-arsenate formation are approximately 9 X 10(-11) M-1 s-1 and 6.3 X 10(-6) M-1 s-1 (Lagunas, R . (1980) Arch . Biochem . Biophys . 205, 67-75). J Mol Biol, 1985 Jun 5, 183(3), 429 - 46 Solution structure of mitochondrial cytochrome c . II . 1H nuclear magnetic resonance of ferrocytochrome c; Moore GR et al.; The 1H nuclear magnetic resonance spectrum of tuna ferrocytochrome c has been studied and the resonances of all 49 amino acid methyl groups have been assigned to specific absorption lines . In comparison with resonance assignments in the ferricytochrome c spectrum, the secondary shifts of resonances of ferrocytochrome c are smaller and the identification of characteristic spin-systems from comparison of spectra from homologous proteins more difficult . For this reason, two-dimensional nuclear magnetic resonance exchange correlated spectroscopy has been used to correlate the assigned resonances of tuna ferricytochrome c with previously unassigned resonances of tuna ferrocytochrome c. J Mol Biol, 1985 Jun 5, 183(3), 409 - 28 Solution structure of mitochondrial cytochrome c . I . 1H nuclear magnetic resonance of ferricytochrome c; Williams G et al.; The 1H nuclear magnetic resonance spectra of tuna and horse ferricytochromes c have been investigated and the resonances of all amino acid methyl groups have been assigned to specific absorption lines . The assignment procedure involves principally the comparison of one-dimensional nuclear magnetic resonance spectra from a range of homologous ferricytochromes c and does not require a prior knowledge of the secondary or tertiary protein structure . Of the 49 methyl groups of tuna cytochrome c, the assignment of 33 is made without reference to the X-ray crystal structure . The method should therefore be applicable to other proteins of similar size where X-ray structures are unavailable . The assignments will be used to investigate the structure of cytochrome c in solution. Cell, 1985 Jun, 41(2), 449 - 56 Isolation of the proto-oncogene c-myb from D . melanogaster; Katzen AL et al.; We have isolated the proto-oncogene c-myb from Drosophila melanogaster . This gene is represented by a single locus at position 13E-F on the X chromosome, and is expressed in early embryos by transcription into two polyadenylated RNAs with lengths of approximately 3.0 and 3.8 kb . The gene may encode a protein with a molecular weight of at least 55,000 that shares a domain with c-myb (chicken) in which 91 of 125 (or 73%) of the amino acids are identical in the Drosophila and chicken genes . These findings represent the first rigorous identification of a Drosophila proto-oncogene that can encode what may be a nuclear protein, and they set the stage for a genetic analysis of how c-myb serves the normal organism. Mol Cell Biol, 1985 Jun, 5(6), 1465 - 72 Structure of the promoter of the Dictyostelium discoideum prespore EB4 gene; Barklis E et al.; EB4 is one of several cloned cDNAs that is expressed as mRNA only after the aggregation stage of Dictyostelium discoideum differentiation and exclusively in prespore and spore cells (E . Barklis and H . F . Lodish, Cell 32:1139-1148, 1983) . We have isolated the unique genome fragment corresponding to the 5' portion of the EB4 message and the EB4 promoter . The EB4 transcript has an unusually long, G + C-rich, 5' noncoding region, but initiates at several start sites within a region of DNA that is 96% A + T . The sequence GTGGTGG, along with slight variations, occurs several times in the promoter . We have used the EB4 promoter to drive the transcription of an EB4/beta-galactosidase fusion transcript in yeast cells . Although the cap sites of the fused transcript in yeast cells are located in the region where multiple EB4 transcripts are initiated in Dictyostelium, the unregulated expression of the fusion transcript in yeast does not mimic the normal regulated pattern of EB4 mRNA expression in D . discoideum. Mutat Res, 1985 Jun-Jul, 150(1-2), 51 - 9 Analysis of non-linearities in mutation frequency curves; Haynes RH et al.; Mutation frequency curves for ultraviolet light and other mutagens often exhibit non-linear, as well as linear components . A common pattern observed for UV-induced reversion of auxotrophy in yeast is a biphasic, linear-quadratic (or higher order) response . The non-linear component in such a biphasic frequency curve can arise in two distinct, but non-mutually exclusive, ways: (i) as a result of the existence of two-hit processes in the molecular mechanism(s) of mutagenesis; and (ii) as a result of the possible stochastic dependence of mutation and killing, such that the probability of clone formation by the mutant cells differs from that of the non-mutant cells in the population . We describe here a simple mathematical method for distinguishing between these two sources of non-linearity . It is based on the calculation of a quantity that we call 'apparent survival.' This is given, for any mutagen dose chi, by the ratio of the mutant yield to the corresponding linear component of mutation frequency . If the apparent survival rises to values greater than unity before declining at high doses, then there must exist positive two-hit (or higher order) components in the mutational mechanism . If the final slope of the apparent survival curve differs from that of the measured survival curve, then there also exists some degree of stochastic dependence between mutation and killing. Mutat Res, 1985 Jun-Jul, 150(1-2), 203 - 10 Genetic change may be caused by interference with protein-protein interactions; Zimmermann FK et al.; Several aprotic polar solvents were shown to induce mitotic aneuploidy in yeast: diethyl ketone, gamma-valerolactone, pyridine, pivalinic acid nitrile, phenylacetonitrile and fumaric acid dinitrile . Only fumaric acid dinitrile also strongly induced other types of genetic effects including mitotic crossing-over, mitotic gene conversion and point mutation . The other substances only induced aneuploidy and this only over a very narrow dose range . The treatment protocol used suggested that these chemicals acted via interference with tubulin assembly and disassembly causing a malfunctioning of spindle fiber microtubules . This hypothesis was tested using twice recycled porcine brain tubulin . Diethyl ketone, gamma-valerolactone, pyridine and phenylacetonitrile inhibited GTP-promoted assembly of porcine brain tubulin in vitro in the concentration range needed for the induction of mitotic aneuploidy in yeast . Pivalinic acid nitrile accelerated tubulin aggregation whereas fumaric acid dinitrile had no effect even at concentrations 18 times higher than the lowest tested concentration effective in yeast . The in vitro experiments with porcine brain tubulin further suggest that genetic change can result from interference with specific protein-protein interactions . Fumaric acid dinitrile was the only exception since it did induce aneuploidy but had no effects on the assembly of porcine brain tubulin . This could be caused either by interference with protein-protein interactions other than between molecules during assembly and disassembly of microtubules or species-specific differences in susceptibility between yeast spindle and porcine brain tubulin. Cell, 1985 Jun, 41(2), 383 - 94 An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene; Jacquier A et al.; The intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae possesses a long internal reading frame (ORF) that is conserved in various yeast species . In crosses between intron-plus and intron-minus variants, this intron determines a specific gene conversion phenomenon, which results in the integration of the intron sequence within all previously intron-minus copies of the gene . We show, from a frameshift mutant within the intron ORF and from the need of mitochondrial protein synthesis, that ORF encodes a protein active in the gene conversion that spreads the intron within populations of interbreeding strains . This new intron function is reminiscent of the "transposase" encoded by mobile genetic elements and is discussed in relation to other intron functions. Biochimie, 1985 Jun, 67(6), 651 - 5 The interferon-induced enzyme 2-5A synthetase adenylates tRNA; Justesen J et al.; The interferon-induced enzyme 2-5A synthetase is shown to adenylate tRNA . Yeast tRNAPhe was incubated with the enzyme in the presence of double stranded RNA (in this case polyI-polyC) and ATP or deoxyATP . The reaction products were analyzed by ribonuclease T1 digestion of the tRNA, polyacrylamide gel electrophoresis and autoradiography . Using ATP, the 2-5A synthetase adds one, two or three AMP residues to the 3'-end of the tRNA whereas when dATP is replacing ATP, only one nucleotide unit is added . It is concluded that one of the mechanisms of the interferon-induced antiviral effect may be an inhibition of the translation process caused by an inactivation of tRNA molecules by a 2-5A synthetase catalyzed 2'-adenylation of the 3'-end. Biochem Biophys Res Commun, 1985 May 31, 129(1), 287 - 92 Stabilization of the adenylate energy charge by the depletion of adenylates without glycolytic stimulation; Yoshino M et al.; A relationship between the AMP deaminase reaction and the recovery of the energy charge was examined using permeabilized yeast cells . Citrate inhibited the glycolytic flux and the recovery of the energy charge . The addition of spermine enhanced the recovery of the energy charge without the reversal of the inhibition of glycolysis in the presence of excess citrate . The AMP deaminase reaction can participate in the stabilization of the energy charge only by the depletion of total adenylates, and not by the glycolytic stimulation under the conditions where citrate is highly increased during aerobic growth conditions. J Biol Chem, 1985 May 25, 260(10), 6133 - 8 On the specificity of cytochrome c synthetase in recognition of the amino acid sequence of apocytochrome c; Visco C et al.; Two forms of yeast cytochrome c synthetases with different specificities were resolved, one (synthetase I), solubilized from mitochondria or the cell debris with Triton X-100, recognizing not horse apocytochrome c but yeast apo-iso-1-cytochrome c as a substrate and the other (synthetase II) still bound with the particulate fraction from mitochondria after treatment with Triton, recognizing both horse and yeast apocytochromes c . The activity with labeled yeast apo-iso-1-cytochrome c as a substrate of cytochrome c synthetase I can be quantitatively inhibited by nonlabeled Candida krusei apocytochrome c and partially by nonlabeled tuna apocytochrome c but not by nonlabeled horse apocytochrome c indicating a specific amino acid sequence being recognized . However, an enzyme similarly solubilized from beef heart mitochondria recognized both horse apocytochrome c and yeast apo-iso-1-cytochrome c for attachment of heme . In view of the fact that the yeast synthetase II and the beef synthetase can both utilize either horse apocytochrome c or yeast apo-iso-1-cytochrome c as substrates, we suggest that these enzymes may also be involved in biosynthesis of cytochrome c1, that is, the ability to attach heme to apocytochrome c and apocytochrome c1 may have been conserved in eucaryotic cells, and that both synthetases may therefore be homologous. Biochim Biophys Acta, 1985 May 20, 829(1), 119 - 26 Trapping of reactive intermediates in enzymology . Exogenous flavin reduction during catalytic turnover of substrate by glyoxalase I; Douglas KT et al.; Although but weak inhibitors of glyoxalase I under steady-state conditions, flavins are reduced by yeast glyoxalase I (lactoyl-glutathione lyase, EC 4.4.1.5) plus its substrate (the hemithiolacetal from glutathione and phenylglyoxal) during catalytic turnover . Studies with 10-ethylisoalloxazine showed that this flavin reduction was peculiar not merely to glyoxalase I's substrate, but was characteristic of the complete system, enzyme plus substrate undergoing catalytic turnover . Flavins are poor hydride-ion acceptors and the reduction observed most likely represents an oxidative trap of a transient carbanion formed in the glyoxalase I mechanism of action . Hydrophobic flavins were more efficient traps than the hydrophilic ones, and values of the Km for the phenylglyoxal: glutathione hemithiolacetal adduct measured by the flavin-reaction and by normal steady-state kinetics were closely similar . This argues that trapping has occurred of an enediolate ion (an enzyme-generated carbanion) still bound to glyoxalase I. Nucleic Acids Res, 1985 May 10, 13(9), 3063 - 82 Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha; Ledeboer AM et al.; The structural gene and the regulatory DNA sequence of the yeast Hansenula polymorpha methanol oxidase have been isolated . According to the nucleotide sequence data obtained, the structural gene encodes a 664 amino acids long protein, contains no intervening sequences, and the 5'- and 3'-non-coding region contains several sequences implicated in transcription initiation and termination in the yeast Saccharomyces cerevisiae . Although the methanol oxidase is translocated to the peroxisomes, no cleavable signal sequence was found at the N-terminus of the protein. Anal Biochem, 1985 May 1, 146(2), 402 - 4 High-performance liquid chromatographic analysis of crystals of tRNA, aminoacyl-tRNA synthetase, and their complex; Lorber B et al.; High-performance liquid chromatography on an ion exchanger column was successfully used for a rapid biochemical analysis of crystals of yeast tRNAAsp and aspartyl-tRNA synthetase as well as cocrystals formed by the synthetase and the tRNA. Cell, 1985 May, 41(1), 31 - 40 A novel ras-related gene family; Madaule P et al.; We have identified a new family of ras genes, the rho genes, which share several properties with the more classical ras gene family consisting of N-, K-, and H-ras . The rho genes, first isolated from a cDNA library from the abdominal ganglia of Aplysia, encode proteins that share 35% amino acid homology with H-ras . Evolutionarily conserved counterparts of rho have been detected in yeast, in Drosophila, in rat, and in man . Sequence analysis reveals over 85% homology between the human and Aplysia proteins . The ras and rho gene products share several common properties; both are 21,000 daltons, both reveal C-terminal sequences required for membrane attachment, and both show blocks of strong internal homology, suggesting that the two proteins may share common functions but may use these functions in different ways. J Cell Biol, 1985 May, 100(5), 1664 - 75 Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme; Schauer I et al.; The role of structural signals in intercompartmental transport has been addressed by the isolation of yeast invertase (SUC2) mutations that cause intracellular accumulation of active enzyme . Two mutations that delay transport of core-glycosylated invertase, but not acid phosphatase, have been mapped in the 5' coding region of SUC2 . Both mutations reduce specifically the transport of invertase to a compartment, presumably in the Golgi body, where outer chain carbohydrate is added . Subsequent transport to the cell surface is not similarly delayed . One mutation (SUC2-s1) converts an ala codon to val at position -1 in the signal peptide; the other (SUC2-s2) changes a thr to an ile at position +64 in the mature protein . Mutation s1 results in about a 50-fold reduced rate of invertase transport to the Golgi body which is attributable to defective signal peptide cleavage . While peptide cleavage normally occurs at an ala-ser bond, the s1 mutant form is processed slowly at the adjacent ser-met position giving rise to mature invertase with an N-terminal met residue . s2 mutant invertase is transported about sevenfold more slowly than normal, with no delay in signal peptide cleavage, and no detectable abnormal physical property of the enzyme . This substitution may interfere with the interaction of invertase and a receptor that facilitates transport to the Golgi body. Immunology, 1985 May, 55(1), 97 - 103 Erythrocyte enhancement of C3b-mediated phagocytosis by human neutrophils in vitro: a combined effect of the erythrocyte complement receptors CR1 and erythrocyte scavengers to reactive oxygen metabolites (ROM); Forslid J et al.; Human erythrocyte CR1 receptors have been shown to bind complement-fixing immune complexes and, thus, facilitate their elimination from the circulation . The autotoxic effect of free radicals released from phagocytes during phagocytosis can be alleviated by scavengers like catalase and superoxide dismutase . Erythrocytes are known to contain these antioxidants . This study showed that 74% of opsonized yeast particles adhered to human erythrocytes . No difference was seen between yeast opsonized with C3b and yeast opsonized with both IgG and C3b . This adherence was due to the C3b receptor (CR1), as monoclonal antibodies against the CR1 receptor could abrogate the adherence . The yeast phagocytosis by neutrophils was increased by 15% when yeast-C3b was used, and by 34% when yeast-IgG/C3b was used in the presence of human red blood cells . The increase of phagocytosis was not seen when rat erythrocytes (lacking CR1) were present . The cytochrome c reduction decreased with the presence of human erythrocytes during phagocytosis, indicating a scavenging effect on the superoxide anions . The addition of scavengers or erythrocyte lysate, instead of erythrocytes, enhanced phagocytosis of yeast-IgG/C3b to at least the same extent as the erythrocytes . These observations suggest that human erythrocytes primarily enhance phagocytosis through the scavenging effect of those erythrocytes which are concurrently attached with the prey through its CR1 receptor, and then attached to the PMN. J Immunol, 1985 May, 134(5), 3307 - 15 Membrane complement receptor type three (CR3) has lectin-like properties analogous to bovine conglutinin as functions as a receptor for zymosan and rabbit erythrocytes as well as a receptor for iC3b; Ross GD et al.; Human leukocyte complement receptor type three (CR3) was shown to be lectin-like and to resemble bovine serum conglutinin (K) in that it bound to both iC3b and unopsonized yeast (Saccharomyces cerevisiae), and was inhibited by EDTA or N-acetyl-D-glucosamine (NADG) . CR3 and K also bound to zymosan (Z), a yeast cell wall extract that contains primarily polysaccharide and no detectable protein . However, structural differences and the absence of K on bovine phagocytes indicated that CR3 was not the human homologue of bovine K . Phagocytic and respiratory responses to unopsonized Z were CR3 dependent because they were inhibited by monoclonal antibodies specific for the alpha-chain of CR3 and did not occur with phagocytes from patients with a genetic deficiency of CR3 . The binding of CR3 to Z did not require opsonization of the Z with neutrophil-secreted C3, as Z binding and responses were not inhibited by Fab anti-C3 . In addition, CR3-dependent binding of yeast occurred with neutrophils from which protein secretion was blocked by fixation with paraformaldehyde . Rabbit erythrocytes (RaE) also bound weakly to neutrophil CR3 and triggered ingestion . Anti-CR3 not only blocked the binding and ingestion of RaE but also blocked selectively the ingestion of RaEC3b without affecting the strong binding mediated by CR1 . Even though sheep E and sheep EC3b were not ingested by neutrophils, a weak binding of CR3 to sheep E was suggested by the finding of 20 to 40% inhibition of sheep EAIgG ingestion by anti-CR3 . Such inhibition was only observed in buffers that allowed activity of the CR3 binding site and not in buffers containing either EDTA or NADG . An apparently contradictory finding was that the weak CR3-dependent binding of Z triggered neutrophil ingestion and a superoxide burst, whereas the avid CR3-dependent binding of sheep EC3bi did not induce significant ingestion or a respiratory burst . Blocking studies with monoclonal antibodies specific for different epitopes of the alpha-chain of CR3 suggested that this might result from the presence of two distinct binding sites in CR3: one site for fixed iC3b that did not trigger functions, and a second function-triggering site for Z that did not bind to fixed iC3b. Acta Virol, 1985 May, 29(3), 254 - 65 Virion proteins and the perspectives of gene manipulations in vaccine preparation; Tickhonenko TI; The achievements and perspectives of genetic manipulations are described aiming at preparation of first generation subunit vaccines based on the synthesis in bacterial and eukaryotic cells of full-sized virion proteins expressing the main antigenic determinants . The preparation of such vaccines in bacterial cells seems out of perspective in the case of influenza, human hepatitis B, foot- and - mouth disease and some other viruses due to the peculiarities of structure and synthesis as well as low immunogenicity of the monomeric form of virion polypeptides . However, biotechnological procedures using eukaryotic cells and higher eukaryotic vectors and in part also yeast cells allowed to obtain full-sized virion proteins in a highly immunogenic state with good effects. J Biol Chem, 1985 Apr 25, 260(8), 5184 - 90 Structure of an electron transfer complex . I . Covalent cross-linking of cytochrome c peroxidase and cytochrome c; Waldmeyer B et al.; Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2 . The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide . The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons . The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase . The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed . The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength . Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues . One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues . The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c. Anal Biochem, 1985 Apr, 146(1), 7 - 12 A colorimetric method for the determination of hydroxamic acid by iodine oxidation; Kobashi K et al.; A new colorimetric method for the determination of hydroxamic acid is described . Hydroxamic acid was oxidized quantitatively by iodine to produce nitrous acid, which was thereafter determined according to the diazocoupling reaction . This method is sensitive to as little as 5 nmol of hydroxamic acid, and the calibration curve is linear up to 50 nmol . Using this method, acyl-CoAs were determined after conversion to hydroxamic acid by the addition of hydroxylamine . The present method is applicable to the determination of free fatty acids which are activated by acyl-CoA synthetase. Mol Cell Biol, 1985 Apr, 5(4), 885 - 9 Characterization and developmental expression of a Drosophila ras oncogene; Mozer B et al.; We cloned a Drosophila melanogaster ras gene (Dmras64B) on the basis of its homology to the ras oncogen from Harvey murine sarcoma virus . This gene mapped at chromosomal position 64B on the left arm of the third chromosome . Sequencing of Dmras64B revealed extensive amino acid homology with the proteins encoded by the human and Saccharomyces cerevisiae ras genes . The coding region of the Drosophila gene is interrupted by two introns located in different positions with respect to its human counterpart . Dmras64B encodes three different RNAs (1.6, 2.1, and 2.6 kilobases long) that are constantly expressed throughout the development of the fly. Anal Biochem, 1985 Apr, 146(1), 125 - 33 Two-dimensional spectrophotometry with high spatial and temporal resolution by digital video techniques and powerful computers; Muller SC et al.; A two-dimensional spectrophotometer having a spatial raster resolution of 512 X 512 picture elements with 256 grey levels and a time resolution of 30 images per min is assembled by combination of digital video techniques and a powerful computer system . The instrument is applied to the analysis of pattern formation processes in cytoplasmic media. Am J Clin Pathol, 1985 Apr, 83(4), 498 - 9 Comparison of media for recovery of clinical isolates of Legionella pneumophila; Keathley JD et al.; Seven media were prepared in house or purchased commercially and were compared for their ability to recover Legionella pneumophila from clinical specimens . Media containing alpha-ketoglutarate from either source detected colonies earlier, more often, and in greater quantities than media without alpha-ketoglutarate . Media without charcoal performed poorly. DNA, 1985 Apr, 4(2), 105 - 14 Genomic clones of Aspergillus nidulans containing alcA, the structural gene for alcohol dehydrogenase and alcR, a regulatory gene for ethanol metabolism; Doy CH et al.; Our aim was to obtain from Aspergillus nidulans a genomic bank and then clone a region we expected from earlier genetic mapping to contain two closely linked genes, alcA, the structural gene for alcohol dehydrogenase (ADH) and alcR, a positive trans-acting regulatory gene for ethanol metabolism . The expression of alcA is repressed by carbon catabolites . A genomic restriction fragment characteristic of the alcA-alcR region was identified, cloned in pBR322, and used to select from a genomic bank in lambda EMBL3A three overlapping clones covering 24 kb of DNA . Southern genomic analysis of wild-type, alcA and alcR mutants showed that the mutants contained extra DNA at sites near the center of the cloned DNA and are close together, as expected for alcA and alcR . Transcription from the cloned DNA and hybridization with a clone carrying the Saccharomyces cerevisiae gene for ADHI (ADC1) are both confined to the alcA-alcR region . At least one of several species of mature mRNA is about 1 kb, the size required to code for ADH . For all species, carbon catabolite repression overrides control by induction . The overall characteristics of transcription, hybridization to ADC1 and earlier work suggest that alcA consists of a number of exons and/or that the alcA-alcR region represents a cluster of alcA-related genes or sequences. Eur J Biochem, 1985 Apr 1, 148(1), 155 - 9 Phosphate dependency of phosphofructokinase 2; Laloux M et al.; Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate . The concentration of inorganic phosphate that allowed half-maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8 . The effect of phosphate was to increase V and to decrease Km for fructose 6-phosphate, without affecting Km for ATP . Citrate and P-enolpyruvate inhibited the enzyme non-competitively with fructose 6-phosphate and independently of the concentration of inorganic phosphate . Phosphorylation of the enzyme by the catalytic subunit of cyclic-AMP-dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate . A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves . By contrast, the fructose 2,6-bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate . Phosphate increased this activity about threefold when measured in the absence of added fructose 6-phosphate and a half-maximal effect was reached at approximately 0.5 mM phosphate . Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect. Biochemistry, 1985 Mar 12, 24(6), 1364 - 76 Mode of interaction of beta-hydroxy-beta-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds; Nakamura CE et al.; The sodium salts of compactin (1) and trans-6-{2-(2,4- dichloro-6-hydroxyphenyl)ethyl}-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran- 2-one (3) are inhibitors of yeast beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase . The dissociation constants are 0.24 X 10(-9) and 0.28 X 10(-9) M, respectively . Similar values have been reported for HMG-CoA reductase from mammalian sources {Endo, A., Kuroda, M., & Tanzawa, K . (1976) FEBS Lett . 72, 323; Alberts, A . W., et al . (1980) Proc . Natl . Acad . Sci . U.S.A . 77, 3957} . The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase . We, therefore, investigated the basis for the strong interaction between HMG-CoA reductase and these inhibitors . HMG-CoA and coenzyme A (CoASH), but not reduced nicotinamide adenine dinucleotide phosphate (NADPH), prevent binding of compactin to the enzyme . HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to the enzyme . We also investigated the inhibitory activity of molecules that resemble structural components of compactin . Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid that is attached to a decalin structure . The sodium salt of DL-3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH . The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 +/- 0.9) X 10(-2) M . Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition . If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be greater than or equal to 0.5 mM . Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition . The sodium salt of (E)-6-{2-(2-methoxy-1-naphthalenyl)ethenyl}-3,4,5,6- tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH . The inhibition constant (vs . HMG-CoA) is 0.8 microM . CoASH does not prevent binding of 6 to enzyme . Compound 6, therefore, behaves analogously to compound 3 . We propose that these inhibitors occupy two sites on the enzyme: one site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1985 Mar 10, 260(5), 2857 - 61 The identification of histidine ligands to cytochrome a in cytochrome c oxidase; Martin CT et al.; A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate {1,3-15N2} histidine into yeast cytochrome c oxidase . Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the {15N}histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome . Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-{15N}imidazole and bis{15N}imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a. J Biol Chem, 1985 Mar 10, 260(5), 2687 - 93 Inhibition of phosphatidylinositol synthase and other membrane-associated enzymes by stereoisomers of hexachlorocyclohexane; Parries GS et al.; Hexachlorocyclohexanes (HCCH) are chlorinated analogs of inositol; the alpha, beta, gamma, and delta isomers of HCCH have the stereochemical configurations of (+/-)-, scyllo-, muco-, and myo-inositol, respectively . To assess their potential as specific tools for the study of agonist-stimulated phosphoinositide metabolism, we examined the effects of these four HCCH isomers on phosphatidylinositol (PI) synthase (CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase), PI:inositol exchange enzyme, and several membrane-associated enzymes unrelated to inositol metabolism . In pancreas microsomes, in the presence of saturating myo-inositol, the alpha, beta, gamma, and delta isomers (4 mM) inhibited PI synthase activity by 9, 4, 22, and 69%, respectively . Half-maximal inhibition by delta-HCCH occurred at 0.25 mM . A similar pattern of HCCH inhibition was obtained using n-octylglucopyranoside-solubilized and partially purified PI synthase preparations . The inhibition by delta-HCCH was noncompetitive versus myo-inositol . The PI:inositol exchange enzyme in mouse pancreas microsomes was inhibited 90% by 1 mM delta-HCCH in the presence of 0.25% Triton X-100, but not in its absence; half-maximal inhibition occurred with 0.5 mM delta-HCCH . delta-HCCH (4 mM) also inhibited to varying extents the following enzymes: pancreas CDP-choline:1,2-diacyl-sn-glycerol cholinephosphotransferase (75%), brain and erythrocyte (Na+,K+)-ATPase (87 and 70%), brain and erythrocyte Mg2+-ATPase (38 and -5%), brain 1,2-diacyl-sn-glycerol kinase (22%), and liver glucose 6-phosphatase (16%) . gamma-HCCH (4 mM) inhibited these enzymes to a lesser extent, or not at all . The order of inhibition by HCCH stereoisomers was the same as the order of their saturation level in phospholipid vesicles (delta greater than gamma greater than alpha greater than beta) . This suggests that the inhibitory action is due to insertion of the compounds either into hydrophobic domains of the enzymes or into annular lipid . The results indicate that the HCCHs are not selective inhibitors of inositol metabolism. Ukr Biokhim Zh, 1985 Mar-Apr, 57(2), 67 - 9 {Histone-specific acetyltransferase from the rat liver nuclei}; Tsudezevich BA et al.; Certain properties of histone-specific acetyltransferases A, B and C, obtained from the rat liver are determined . pH optimum for enzyme A is within the range of 7.5-8.5, for B--7.8 and for C--7.5 . The maximal activity for enzymes A, B and C is observed with the 60 micrograms/ml concentration of the substrate . The activity is inhibited by N-maleimide, iodacetamide and chloromercuribenzoate . The results obtained show that a number of similar properties are typical of the above enzymes. J Inorg Biochem, 1985 Mar-Apr, 23(3-4), 177 - 82 Helical packing in the hydrophobic sector of cytochrome c oxidase; Bisson R et al.; An arrangement for the membrane-spanning segments of the three larger subunits of cytochrome c oxidase is proposed on the basis of sequence comparison and polarity distribution estimated from the data available for 11 different organisms. Biokhimiia, 1985 Mar, 50(3), 485 - 94 {Polymorphism of membrane proteinases from mitochondria.}; Zubatov AS et al.; Using native polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate, the detergent extracts of sonic submitochondrial particles (SMP) were separated into three protein fractions capable of accomplishing the proteolysis of cytochrome c and three other fractions catalyzing the hydrolysis of N-a-benzoyl-L-arginine-p-nitroanilide (BAPA) and N-a-benzoyl-L-arginine-B-naphthylamide (BANA) . The fractions isolated from the gel were subjected to a thorough anaylsis . Cytochrome c hydrolases were shown to have identical molecular weights (17000) but different isoelectric points (4.0, 4.2 and 4.4) . The total cytochrome c hydrolase activity of these enzymes was inhibited by phenylmethylsulfonylfluoride but was insensitive to ethylenediaminetetraacetate and o-phenanthroline . Three BANA (BAPA) hydrolases have identical Mr values (approximately 17500) but different pI values (4.2, 4.3 and 4.7) . Apart from the indicated hydrolases, the detergent extracts of SMP were shown to contain minor components with identical activities distinguished by the tightness of binding to the inner mitochondrial membrane, Mr and sensitivity to proteinase inhibitors . The observed phenomenon is considered to be due to the polymorphism of proteinases coupled with the inner mitochondrial membrane. Cell, 1985 Mar, 40(3), 491 - 500 Ty elements transpose through an RNA intermediate; Boeke JD et al.; We have followed Ty transposition with a donor Ty element, TyH3, whose expression is under the control of the GAL1 promoter . Sequence analysis reveals dramatic structural differences in TyH3 before and after transposition . If the donor TyH3 is marked with an intron-containing fragment, the intron is correctly spliced out of the Ty during transposition, suggesting that the Ty RNA is the intermediate for transposition . Furthermore, the pattern of sequence inheritance in progeny Ty insertions derived from the marked Ty follows the predictions of the model of retroviral reverse transcription . Comparison of marked Ty elements before and after movement shows that transposition is highly mutagenic to the Ty element . These results demonstrate that during transposition, Ty sequence information flows from DNA to RNA to DNA. Biochem J, 1985 Feb 15, 226(1), 275 - 82 Reversible inactivation by noradrenaline of long-chain fatty acyl-CoA synthetase in rat adipocytes; Hall M et al.; Incubation of rat adipocytes with the same range of noradrenaline concentrations that stimulate lipolysis caused a rapid and stable decrease in the activity of fatty acyl-CoA synthetase . Corticotropin, glucagon and dibutyryl cyclic AMP also decreased the activity of the enzyme . The effect of noradrenaline was apparent over a wide range of concentrations for the three substrates of the enzyme . A novel fluorescence assay of fatty acyl-CoA synthetase using (1,N6-etheno)-CoA is described . The effect of noradrenaline was not abolished by inclusion of albumin in homogenization buffers, persisted through subcellular fractionation and isolation of microsomes (microsomal fractions) and even survived treatment of microsomes with Triton X-100 . The effect of noradrenaline was rapidly reversed within cells by the subsequent addition of insulin or propranolol . The inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline . Additions of cyclic AMP-dependent protein kinase to adipocyte microsomes caused considerable phosphorylation of microsomal protein by {gamma-32P}ATP, but did not affect the activity of fatty acyl-CoA synthetase. J Biol Chem, 1985 Feb 10, 260(3), 1407 - 12 Reversible acidic-alkaline transition of the carbon monoxide complex of cytochrome c peroxidase; Iizuka T et al.; The Soret absorption band of the ferrous carbon monoxide (CO) complex of cytochrome c peroxidase exhibited a blue shift from 423.7 to 420 nm upon an increase in pH from 6.5 to 8.5 . The spectral change was reversible with an isosbestic point at 422 nm . The pH dependence of this spectral change gave a sigmoidal curve fitted well to a theoretical curve of a cooperative release of two protons with a pK value of 7.5, indicating the existence of the acidic and alkaline forms of the ferrous CO enzyme . Upon irradiation of light flash (100 J of power and 30-microseconds), the heme-bound CO was readily dissociated in both acidic and alkaline forms with a quantum yield of approximately unity . On the other hand, the rate of recombination of the dissociated CO with the heme iron was significantly different between these two forms; the recombination rate constants were 1.1 X 10(3) and 3.0 X 10(4) M-1 S-1 at 25 degrees C for the acidic and alkaline forms, respectively . At intermediate pH values, kinetics of recombination were biphasic, consisting of the slow and fast processes with the appropriate rate constants mentioned above . When the fraction of the fast process was plotted against pH, the pH profile coincided with the spectrophotometric pH titration curve described above . Thus, it was concluded that the acidic and alkaline forms of the enzyme were responsible for the slow and fast processes, respectively . In infrared spectroscopy, the acidic form showed a narrow CO stretching band at 1922 cm-1 with a half-band width of 12.5 cm-1, while the alkaline form exhibited a broad CO-stretching band at 1948 cm-1 with a half-band width of 33 cm-1 . Significance of these results are discussed in relation to the structure of the heme vicinity on the CO complex of cytochrome c peroxidase. Biochim Biophys Acta, 1985 Feb 8, 833(2), 239 - 44 Substrate specificity of fatty-acyl-CoA ligase in liver microsomes; Noy N et al.; The substrate specificity of fatty-acyl-CoA ligase in liver microsomes has been studied in a system in which fatty acids are present initially as complexes with unilamellar vesicles of phosphatidylcholine . The latter were prepared by cosonication of phospholipids and different fatty acids . As compared with previous studies of the enzyme the activity of acyl-CoA ligase is several-fold higher for assays carried out with fatty acid substrates added as components of a bilayer . This was true for all fatty acids studied . Also as compared with data reported previously in the literature there was a systematic relationship between the structure of fatty acids, activity at Vmax for synthesis of acyl-CoA and avidity of binding to the ligase . Activity at Vmax was greatest for lauric acid and decreased with increasing chain length . The apparent avidity of enzyme for fatty acids was greatest for octanoic acid and decreased as chain length increased. Biochim Biophys Acta, 1985 Feb 4, 827(2), 183 - 9 Physicochemical characterization of the alkaline denaturation of cytochrome c peroxidase; Dowe RJ et al.; The alkaline denaturation of cytochrome c peroxidase and apocytochrome c peroxidase was investigated by analytical ultracentrifugation, gel-filtration chromatography, and circular dichroism . The