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| Biochemistry, 1975 Jul 29, 14(15), 3451 - 8 Products obtained after in vitro reaction of 7,12-dimethylbenz{alpha}anthracene 5,6-oxide with nucleic acids; Blobstein SH et al.; Several lines of evidence suggest that oxide derivatives of carcinogenic polycyclic hydrocarbons are the reactive intermediates for in vivo binding to cellular nucleic acids . In the present study the covalent binding of 7,12-dimethylbenz{alpha}anthracene 5,6-oxide to synthetic homopolymers and nucleic acids in aqueous-acetone solutions has been investigated . Poly(G) was found to be the most reactive nucleic acid and underwent approximately 7-10% modification . Alkaline hydrolysis of the poly(G)-dimethylbenzathracene conjugate yielded chromatographically distinct polycyclic hydrocarbon-modified nucleotides which were further characterized by spectral analyses and enzymatic and chemical degradation . When the oxide was allowed to react with GMP or dGMP, at least two products were obtained in about 1% yield . Acid hydrolysis of the dGMP-dimethylbenzanthracene conjugates liberated the corresponding guanine-dimethylbenzathracene products . Mass spectral analysis of the modified bases provided direct evidence that we had obtained covalent binding of the poly-cyclic hydrocarbon to guanine . The mass spectral cleavage pattern suggest that one of these products is a hydroxydihydro derivative of dimethylbenzanthracene bound to guanine and the other is a dimethylbenzanthracene-guanine conjugate . Additional structural aspects of these guanine derivatives are discussed. Biochim Biophys Acta, 1975 Jul 23, 395(4), 455 - 67 Partial degradation of transfer RNAs by different preparations of snake venom exonuclease; Petrova M et al.; The degradation of yeast tRNASer with eight different exonuclease preparations from four snake venoms was investigated . The reaction products were separated on polyacrylamide gels containing 7 M urea . Patterns of sharp bands were obtained which were more or less similar . Two tRNA fragments were characterized by oligonucleotide analyses, one of which was tRNA degraded by the exonuclease up to the beginning of the T-phi-C-stem . The other one was generated by the additional loss of several nucleotides from the 5'-terminus . The formation of the latter fragment was very probably caused by an endonuclease activity in the exonuclease . The endonuclease contaminant, which was found in all preparations, was further investigated by experiments with modified tRNAs whose 3'-terminus should be resistant to exonuclease (tRNASer-A, tRNASerOX-red) . With 3'-AMP as substrate no phosphatase activity was found under the conditions of tRNA degradation . Not only in tRNASer, but also in yeast tRNATyr and tRNAAla as well as in fragments of tRNASer and tRNAPhe, the degradation by exonuclease was inhibited at the beginning of the T-phi-C-stem . The finding of such a retardation site in addition to the general retardation of exonuclease digestion after removal of the C-C-A sequence may indicate that retardation at certain elements of secondary structure is a more general feature of degradations by this enzyme. J Biol Chem, 1975 Jul 10, 250(13), 5221 - 6 Evidence for an essential histidine in carboxypeptidase Y . Reaction with the chloromethyl ketone derivative of benzyloxycarbonyl-L-phenylalanine; Hayashi R et al.; The possible role of histidine residues in the catalytic function of carboxypeptidase Y from bakers' yeast has been investigated using site-specific reagents . Among the reagents tested, benzyloxy-L-phenylalanylchloromethane (Z-PheCH2Cl) was the most powerful inhibitor of the enzyme . It irreversibly inactivated both the peptidase and esterase activities with an apparent second order rate constant of 3.8 M-minus 1 S-minus 1; the D isomer caused essentially no effect on either activity . Inhibition by L-Z-PheCH2Cl, the reaction retarded by certain competitive inhibitors of the enzyme . Using radioactive L-Z-PheCH2Cl, the reaction with the enzyme was shown to be essentially stoichiometric . Diisopropylphosphorofluoridate (iPr2PF)-inactivated enzyme failed to react with Z-PheCH2Cl, and conversely, the Z-PheCH2Cl-inhibited enzyme failed to react with radioactive iPr2PF . Amino acid analyses of the Z-PheCH2Cl-inactivated enzyme revealed the loss of essentially 1 residue, with a concomitant yield of a 0.62 residue of N-t-carboxymethylhistidine . Since carboxypeptidase Y has a reactive serine at its active center, we concluded from these results that the mechanism involves a charge-relay system in the hydrolysis of peptide and ester substrates, as in chymotrypsin . An -SH group of carboxypeptidase Y was not affected during the reaction with L-Z-PheCH2Cl . The generic name "serine carboxypeptidase" has been proposed for carboxypeptidase Y and for the iPr2PF-sensitive carboxypeptidases from plants, molds, and animal tissues, in order to distinguish them from "metal carboxypeptidase" to which carboxypeptidase A (EC 3.4.12.2) and B (EC 3.4.12.3) belong. Biosystems, 1975 Jul, 7(1), 137 - 46 The response of oscillating glycolysis to perturbations in the NADH/NAD system: a comparison between experiments and a computer model; Richter O et al.; The glycolytic pathway is described by a set of coupled non linear differential equations of first order with respect to time . The individual terms of these equations consist of enzyme velocities assuming a steady state hypothesis for the enzymatic forms . These are specified and the system is solved numerically . Oscillations are explained by interaction of PFK with the adenylate system . The conditions for the occurrence of oscillations are tested in a series of computer runs . The phase relations between intermediates of the model agree with those found in yeast cells . As an application of the model the disturbation of oscillations by the addition of acetaldehyde is simulated . The predictions of the model agree with experimental results. Biochim Biophys Acta, 1975 Jun 2, 395(1), 28 - 40 The isolation of large polysomes in high yield from unfractionated tissue homogenates; Morton B et al.; It was found that if large quantities of both exogenous RNA and Mg-2+ were present during gentle tissue homogenization, the subsequent addition of deoxycholate to the whole homogenate produced a viscous mass from which polysomes could be isolated in large yields . These polysomes were substantially less degraded than those isolated by previous methods . In the case of rat liver, 15 ribosomes per mRNA was the species present in highest concentration . The parameters of this method were investigated and optimized . About 80 percent of the rRNA in the homogenates was recovered in the polysomes . Omission of deoxycholate permitted the isolation of less-degraded free polysomes as well . In the liver of fed rats these represented one-fourth of the total polysomes, in good agreement with results obtained by an independent approach . Using the method to isolate polysomes from the liver of starving rats, it was found that only about one percent of the large amount of monomers and dimers present resulted from polysome breakdown during isolation . It was further shown that random RNAase hydrolysis of polysomes could not produce the patterns of liver polysomes seen during starvation . Polysomes isolated by this procedure were quite stable in solution and were very active in cell-free protein synthesis . Application of this method without adaptation to eight other tissues also permitted the isolation of large polysomes in high yields. Eur J Biochem, 1975 Jun, 54(2), 549 - 66 Flavocytochrome b2: kinetic studies by absorbance and electron-paramagnetic-resonance spectroscopy of electron distribution among prosthetic groups; Capeillere-Blandin C et al.; The reduction by L-lactate of the prosthetic groups of flavocytochrome b2 (L-lactate cytochrome c oxidoreductase from aerobic yeast, a tetrameric molecule containing one haem and one flavin mononucleotide per protomer) was reinvestigated . It was confirmed that the enzyme ultimately takes up 3 electrons per protomer from this 2-electron donor . Stopped-flow absorbance data at an haem isosbestic point to follow the oxidized flavin and in a haem band indicate that, under the conditions used, haem and flavin reduction time courses are indistinguishable, both being biphasic (phases I and II) . Comparison with electron paramagnetic resonance data (Fe3+ haem and flavosemiquinone signals) led to a complete description at 24 degrees C of the time courses of the various reduction states of the prosthetic groups . It has been previously demonstrated (Morton and Sturtevant, 1964) that, after the formation of the enzyme-substrate complex, the electron transfer to the enzyme takes place as the first and rate-limiting step of the turnover . In the present study, an initial burst of fully reduced flavin, of small amplitude, is detected at the very beginning of phase I (before 6 ms) . The redox forms which accumulate thereafter till the end of phase I (30-35 ms) are the reduced haem (up to 80%), the flavin semiquinone (up to 50%) and the fully reduced flavin (from 25% up to 35%); the total of electrons distributed at the end of phase I is about 2 per protomer meaning that, in this phase, each enzyme site acts as a 2-electron and not a 3-electron acceptor . A 2-electron flow as the limiting step during phase I with the rate constant kI accounts for the steady-state electron flow during catalysis . Phase I is followed by the much slower phase II which corresponds to the entry of the third electron and cannot be involved in the turnover . The interpretation of the results are given as a scheme, with the proper rate constants, allowing a satisfactory fitting of experimental data by simulation . Among the elementary steps required are a rapid distribution of one electron from reduced flavin to the haem, a rapid interprotomers dismutation between couples of flavin semiquinone regenerating two oxidized flavin per tetramer . The very low reactivity of the latter for the entry of the third electron per protomer is tentatively explained by the occurrence of a slow additional step limiting the final reduction reaction . It was observed that, over phase I and the beginning of phase II, from 15 to 200 ms, all the redox species remain apparently under equilibrium conditions . Parallel studies (titrations of flavocytochrome b2 by L-lactate) showed that the set of equilibrium parameters relative to haem and flavin species is significantly different in the "final" equilibrium (after 30 s) from that in the time interval 15-200 ms . Such an anomaly suggests a conformation change takes place very slowly in the molecule after the acceptance of the first two electrons per protomer. J Biochem (Tokyo), 1975 Jun, 77(6), 1313 - 8 Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue; Hayashi R et al.; The metal content of carboxypeptidase Y was analyzed by the atomic absorption method . After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co) . The activity was, however, rather sensitive to preincubation with various metals . The reactivity of a serine residue of the enzyme was also reevaluated . Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme . The rate of inactivation with DFP was much faster than that for typsin {EC 3.4.21.4} and chymotrypsin {EC 3.4.21.1.}, while the rate with PMSF was one-fifteenth of that for chymotrypsin . The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester . The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified. J Biol Chem, 1975 May 25, 250(10), 3785 - 9 Glycerol as an agent eliciting small conformational changes in alcohol dehydrogenase; Myers JS et al.; Yeast alcohol dehydrogenase is an example of a protein in which the K-m for substrate is substantially decreased by the presence of glycerol . The polyol has the effect at pH 8.0 or above of decreasing K-m and K-s for substrate and of altering both the protein's intrinsic fluorescence and ultraviolet absorption difference spectrum . The relationship between each of thse parameters and glycerol concentration displays a transition at a glycerol concentration of 20% . Circular dichroism values for the enzyme are not affected by glycerol over a large range of concentration and temperature . Treatment of the enzyme with glutaraldehyde results in the formation of cross-linked tetramers, the K-m of which are not altered by the presence of the solvent . The data are interpreted as reflecting a change in the conformation of the protein induced by glycerol. Eur J Biochem, 1975 May 6, 53(2), 533 - 9 Nuclease digestion of synthetase x tRNA complexes; Horz W et al.; Phenylalanyl-tRNA and seryl-tRNA synthetase protect strongly though not completely their cognate tRNAs against nuclease attack, as had been shown previously . In an investigation of the mechanism of protection it was demonstrated that the low susceptibility of phenylalanyl-tRNA-synthetase x tRNA-Phe complexes to nucleases is due to free tRNA present in equilibrium with synthetase . The equilibrium can be shifted by an excess of synthetase or by dilution of the complex . It therefore appears that synthetase competes with the nuclease for free tRNA . Degradation of the complex is low, however, because under the conditions of partial digestion the synthetase has a greater affinity for the tRNA than does the nuclease . Fragmented tRNAs, as they are formed during partial nuclease digestion, bind to synthetase to different degrees . tRNA-Phe with a lesion in the dihydrouridine loop binds very poorly whereas a nick in the anticodon loop reduces the strength of binding to a much lesser extent . In a systematic study of the stoichiometry of protection it was confirmed that under standard conditions one phenylalanyl-tRNA synthetase protects one tRNA-Phe and one seryl-tRNA synthetase two tRNA-Ser molecules against nuclease attack . Under certain conditions, however, (concentration of the complex higher than 10 mu-M, or alternately in buffers of low ionic strength) it is observed that phenylalanyl-tRNA synthetase binds up to 1.6 molecules tRNA-Phe . In the serine system, these special conditions do not affect the binding properties of seryl-tRNA synthetase. Proc Natl Acad Sci U S A, 1975 May, 72(5), 1940 - 4 Presence of two polypeptide chains comprising fatty acid synthetase; Stoops JK et al.; Highly purified fatty acid synthetases of chicken and rat livers have molecular weights of 500,000 and dissociate in solutions of low ionic strength into subunits of molecular weight 250,000 with loss of synthetase activity . The subunits can be reassociated in phosphate buffer with full restoration of the activity . In the presence of sodium dodecyl sulfate or guanifine-HCl, the synthetases dissociate into polypeptide chains of molecular weight 220,000 as determined by sodium dodecyl sulfate-gel electrophoresis and sedimentation equilibrium . The polypeptide contains the 4-phosphopantetheine group and the {14C}acetyl and {4C}malonyl groups if the synthetases were prelabeled with {14C}acetyl-CoA and {14C}malonyl-CoA . Similar results were obtained with the synthetase from yeast, except the subunit has a molecular weight of 200,000 . These observations indicate that the multi-catalytic activities of the synthetases and the acyl carrier protein are associated only with the two polypeptide chains . The findings suggest a novel structural organization for multienzyme complexes. Proc Natl Acad Sci U S A, 1975 May, 72(5), 1887 - 91 Single-strand scissions of chromosomal DNA during commitment to recombination at meiosis; Jacobson GK et al.; Diploid cells of the yeast Saccharomyces cerevisiae induced to undergo meiosis accumulate single-strand scissions in both template and newly synthesized DNA during commitment to genetic recombination . No evidence for accumulation of double-strand breaks during meiosis was obtained . When commitment to recombination is at the full meiotic level there are approximately 70 to 200 single-strand scissions per meiotic cell in which approximately 150 recombination events have been reported to occur. J Dairy Sci, 1975 May, 58(5), 668 - 71 Pimaricin and mycostatin for retarding cottage cheese spoilage; Nilson KM et al.; Two antifungal agents, pimaricin and mycostatin, added to Cottage cheese through the wash water at concentrations of 20, 50, or 100 mug/ml of wash water or added through the cheese dressing at 1, 2, or 5 mug/g retarded the growth of Aspergillus niger and Saccharomyces cerevisiae and improved the shelf-life of the cheese . In general, cheese with highest concentration of antifungal agent and stored at lowest temperature had best keeping quality . Pimaricin was slightly more effective than mycostatin in inhibiting fungi; inhibition was greater if the antifungal agents were added to the cheese dressing and the cheese was stored at low temperature; and A . niger was more sensitive to the inhibitors than S . Cerevisiae. Genetics, 1975 May, 80(1), 77 - 85 Mutation of a heterothallic strain to homothallism; Hopper AK et al.; Upon mutagenesis, a heterothallic alpha-alpha diploit strain mutated to homothallism . The gene confering homothallism is nuclear, recessive, and unlinked to mating type . This gene is not allelic to the HO gene, which is responsible for previously described instances of homothallism in yeast . We have designated this new gene for homothallism as cmt (change of mating type). J Med Chem, 1975 May, 18(5), 530 - 3 2-Phenethylimidazole derivatives . Synthesis and antimycotic properties; Godefroi EF et al.; Compounds of type (X = O, NH; Ar and Ar' = phenyl of substituted phenyl; ten examples) were prepared and assayed against miconazole (II,X = O; Ar = Ar' =2,4-Cl2C6H3) as potential antimycotic agents . Optimal activity was noted for I(X = O; Ar = Ar' = 2,4-Cl2C6H3), the direct analog of miconazole . It is about one-tenth as active. Biochim Biophys Acta, 1975 May 1, 390(2), 231 - 45 Nucleotide regulation of a eukaryotic protein synthesis initiation complex;; Walton GM et al.; Formation of a ternary initiation complex containing Met-tRNAf, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex . The concentration of GTP required for half maximal formation of the ternary complex is 2.5 with 10(-6) M . GDP is a potent competitive inhibitor of ternary complex formation with Ki = 3.4 with 10(-7) M . The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with KD(GDP) = 3.0 with 10(-8) M; 100-fold higher concentrations of GTP than GDP are required for displacement of either {(3)H}GDP or {(3)h}gtp from the necleotide binding site . An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP . Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis. Biochim Biophys Acta, 1975 Apr 19, 384(2), 331 - 41 On the role of tryptophan residues in the mechanism of action of glyceraldehyde-3phosphate dehydrogenase as tested by specific modification; Heilmann H-D et al.; A method is described to selectively modify one of the three tryptophan residues of the subunit of glyceraldehyde-3-phosphate dehydrogenase from yeast . As modifying agent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide was used . The residue which is modified by the procedure described has been identified as Trp-193 . There are either one or two molecules of the modifying agent being added to this tryptophan side chain . The modification apparently does not cause a detectable conformational change of the protein as judged from the methods employed . However, the enzymatic activities in the dehydrogenase as well as in the esterase reactions are lost after the modification . It could be established that the modification rendered the enzyme unable to bind the oxidized coenzyme . Also the charge-transfer interaction between enzyme and coenzyme could no longer be observed. Biochim Biophys Acta, 1975 Apr 7, 385(2), 312 - 23 The S-n-propyl analogue of S-adenosylmethionine; Schlenk F et al.; A special strain of Saccharomyces cerevisiae responded to a supplement of S-n-propyl-L-homocysteine in the culture medium by synthesizing S-adenosyl-(S-n-propyl)L-homocysteine, the S-n-propyl analogue of S-adenosylmethionine . S-n-Butyl-L-homocysteine reacted sparingly with this strain, but S-isopropyl-L-homocysteine failed to form detectable quantities of the corresponding S-adenosylsulfonium compound . The S-n-propyl compound was isolated by extraction of the cells, followed by ion-exchange chromatography, which separated it from endogenous S-adenosylmethionine . The structure was determined by hydrolytic procedures leading to overlapping fragments of known structure, 5'-n-propylthioadenosine and S-n-propyl-L-homocysteine . The new sulfonium compound was examined for its activity as n-propyl donor by substituting it for S-adenosylmethionine in methyltransferase systems . Enzymatic transpropylation was observed with S-adenosylmethionine : L-homocysteine S-methyltransferase (EC 2.1.1.10) . Its rate was low in the S-adenosylmethionine : N-acetylserotonin O-methyltransferase system (EC 2.1.1.4), and below recognition with S-adenosylmethionine : guanidinoacetate methyltransferase (EC 2.1.1.2) and S-adenosylmethionine : histamine N-methyltransferase (EC 2.1.1.8). Arch Dis Child, 1975 Apr, 50(4), 311 - 7 Familial opsonization defect associated with fatal infantile dermatitis, infections, and histiocytosis; Scott H et al.; Members of four generations of a family had a defect of serum opsonization for yeast phagocytosis consistent with dominant inheritance . 2 were healthy, one had chronic osteomyelitis, and the fourth developed a fatal illness in infancy characterized by exfoliative dermatitis, diarrhoea, multiple bacterial infections, and failure to thrive, which resembled the two prevously reported cases with this opsonization defect . At necropsy the infant also had lymphoid depletion, which was possibly secondary, and massive histiocytic infiltration. Biochim Biophys Acta, 1975 Mar 28, 386(1), 365 - 8 Essential arginine residues in D-glyceraldehyde-3-phosphate dehydrogenase; Nagradova NK et al.; Two arginyl residues per subunit of yeast D-glyceraldehyde-3-phoshphate dehydrogenase were modified by treatment with butanedione without significant changes in the compostion of other amino acid residues . The modified enzyme displays no dehydrogenase activity . It retains the capacity for interacting with the coenzyme NAD, but binds it less firmly than does the native enzyme . The molar absorbance of the enzyme-NAD complex is markedly reduced and the reactivity of the active-center SH groups is changed in the modified enzyme . The native and modified enzymes show identical fluorescence spectra, absorbance and CD spectra. Biochemistry, 1975 Mar 11, 14(5), 963 - 9 Interaction of steroids with nucleic acids; Arya SK et al.; 17beta-Estradiol and testosterone bind to both native and denatured DNA, and to RNA and poly(A)-poly(U) . Binding affinity depends on the conformation of nucleic acid . Lowering the electrolyte concentration and raising the temperature increase the binding of 17beta-estradiol to native DNA and decrease that to denatured DNA . In 0.01 M NaCl and at 37 degrees, more 17beta-estradiol is bound to native DNA than to denatured DNA . Higher binding of steroid to denatured DNA relative to native DNA at low temperature and high ionic strength is related to larger fraction of binding sites per unit nucleotide in denatured DNA . In addition to 17beta-estradiol and testosterone, 17alpha-estradiol, 17beta-estradiol-3-methyl ether and 19-nortestosterone also stabilize the structure of nucleic acids and poly(A)-poly(U) against thermal denaturation . The 17beta-estradiol induced elevation of the T-m of DNA is diminished by methanol or high NaCl concentration . These results indicate the involvement of hydrogen bonding and hydrophobic interactions between steroids and nucleic acids . The results of binding isotherms and optical studies suggest a conformational dependence of the binding of steroids to nucleic acids. Biochemistry, 1975 Mar 11, 14(5), 981 - 8 Ribonucleoside 3'-di- and -triphosphates . Synthesis of guanosine tetraphosphate (ppGpp); Kozarich JW et al.; A procedure has been outlined for the synthesis of ribonucleoside 3'-di- and -triphosphates . The synthetic scheme involves the conversion of a ribonucleoside 3'-monophosphate to its 2'-(5'-di)-O-(1-methoxyethyl) derivative, followed by successive treatments of the blocked ribonucleotide with 1,1'-carbonyldiimidazole and mono(tri-n-butylammonium) phosphate or pyrophosphate . The resulting ribonucleoside 3'-di- and -triphosphate derivatives are then deblocked by treatment with dilute aqueous acetic acid, pH 3.0 . The use of this procedure is illustrated for adenosine 3'-monophosphate, which has been converted to its corresponding 3'-di- and -triphosphates in 61% overall yield . The decomposition of adenosine 3'-di- and -triphosphates to adenosine 2'-monophosphate, adenosine 3'-monophosphate, and adenosine cyclic 2',3'-monophosphate as a function of pH at 100 degrees has been studied as has the attempted polymerization of adenosine 3'-diphosphate with polynucleotide phosphorylase . Also prepared was guanosine 5'-diphosphate 3'-diphosphate (guanosine tetraphosphate; ppGpp), which was accessible via treatment of 2'-O-(1-methoxyethyl)guanosine 5'-monophosphate 3'-monophosphate with the phosphorimidazolidate of mono(tri-n-butyl ammonium) phosphate . The resulting blocked tetraphosphate was deblocked in dilute aqueous acetic acid to afford ppGpp in an overall yield of 18%. Arch Microbiol, 1975 Mar 10, 102(3), 293 - 8 Cytochemical detection of polysaccharides on the surface of the cell membrane complex in fungi; Vorisek J et al.; Cytochemical staining in toto (periodic acid, thiosemicarbazide, OSO4) revealed the presence of polysaccharide lamellae on the surface of the cell membrane complex of fungi . The membraneous clusters in the vacuolar bodies of Claviceps purpurea were covered with these lamellae at both surfaces, as it was also the case with the endoplasmic reticulum membranes, the tonoplast and the cytoplasmic membrane . In Saccharomyces cerevisiae, the polysaccharide lamellae were visible on the surface of the endoplasmic reticulum membranes and the plasmalemma; the strain revealed polysaccharide deposits also on the tonoplasts of small vacuoles and in glucanase vesicles . We assume that these observations give precision to the localization of the enzymes synthetizing the glycoprotein components of the fungal cell wall. J Biol Chem, 1975 Feb 25, 250(4), 1413 - 21 Radiolabeling of proteins and viruses in vitro by acetylation with radioactive acetic anhydride; Montelaro RC et al.; We describe a convenient, rapid, and reproducible method for labeling proteins in vitro by acetylation with {3H} or {14-C}acetic anhydride dissolved in small amounts of anhydrous dioxane . The reaction is carried out at neutral pH and does not require the use of detergents, water-immiscible organic solvents, oxidizing, or reducing agents . Thus undesirable solvent-induced alterations in protein structure and biological activity are minimized . A method for calculating the specific activity of the protein and the efficiency of acetylation at known concentrations of protein and acetic anhydride is presented . Radioacetylated proteins were shown to be suitable for use as molecular weight calibration standards and as protein markers in polyacrylamide gel electrophoresis, gel filtration, and enzyme studies . Acetic anhydride was used to label intact oncornaviruses, which consist of a complex ribonucleo-protein core within a lipid envelope . Some of the viral lipid and all of the viral proteins, including the internal ones, were labeled without detectable alterations in viral morphology or buoyant density . This result suggests that acetic anhydride, evidently by virtue of its small size and neutral charge, penetrates freely throughout the viral membrane and core structures . The reactivity of RNA with acetic anhydride was less than 1% that of protein under similar reaction conditions. Eur J Biochem, 1975 Feb 21, 51(2), 403 - 13 Studies on energy-linked reactions: genetic analysis of venturicidin-resistant mutants; Lancashire et al.; Genetic analysis of venturicidin-resistant mutants has revealed the presence of both nuclear and mitochondrial genes responsible for determining venturicidin sensitivity/resistance in Saccharomyces cerevisiae . Recombination studies show that the mutation with phenotype VENR is situated at mitochondrial locus OL I and is therefore extremely useful of future genetic manipulations as it gives a unique phenotype to this locus distinguishable from the second oligomycin locus OL II . The mutations with phenotype VENR OLYR are linked to oligomycin locus OL I and have been allocated a new mitochondrial locus, namely OL III . Three factor croses involving the venturicidin mutations at loci OL I and OL III have shown them to freely recombine with the other mitochondrial loci R I, R III and OL II . The mitochondrial genetic map is therefore represented as four 'recombinational linkage groups' . A fifth linkage group is also specified for mutants with phenotype VENR TETR, and is probably located on a separate DNA molecule from the four other groups. Biochim Biophys Acta, 1975 Feb 13, 381(2), 301 - 7 Uridine diphosphate 2-deoxyglucose . Chemical synthesis, enzymic oxidation and epimerization; Druzhinina TN et al.; The paper describes chemical synthesis of uridine diphosphate 2-deocyglucose (UDPdGLc) through reaction of uridine 5'-phosphomorpholidate with 2-deoxy-a-D-glucopyranosyl phosphate . The prepared analog of uridine diphosphate glucose (UDPGlc) served as a substrate for calf liver UDPGlc dehydrogenases (EC 1.1.1.22), the reaction product was identified as nucleotide deoxyhexuronic acid derivative . The apparent Km for UDPdGlc was found to be 60 times that of UDPGlc, and the relative V value for the analog was 0.09 . The peculiar lag-eriod in reaction kinetics has been observed for the analog and is presumably connected with the slow rate of the initial stages of the reaction . UDPdGlc was found to be quite an efficient substrate for UDPGlc 4-epimerases (EC 5.13.2) from yeast, calf liver and mung bean seedlings. Biophys Chem, 1975 Feb, 3(1), 90 - 8 High pressure effects on the activity of glycolytic enzymes; Schmid G et al.; High hydrostatic pressure inhibits growth in most organisms; this may be explained by a deactivation of enzymes involved in essential metabolic pathways . In order to check this hypothesis the enzymic activity of rabbit muscle lactic dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase was investigated in the presence of the coenzyme and excess of substrate at pressures up to 2kbar . Kinetic analysis of an initial phase of pressure induced activation and of a second phase of reversible deactivation shows that the two enzymes respond to high pressures in different ways leading to a volume of activation of increment V is not equal to (LDH) equal 0 plus or minus 1 cm-3 mol-1 and increment V is not equal to (GAPDH) equals 60 plus or minus 4 cm-3 mol-1, respectively . Comparing the lower limits of pressure deactivation, LDH is found to be more stable towards pressure than GAPDH . At p is approximately equal to 2 kbar total deactivation of both enzymes is observed . A concentration dependent lag of GAPDH reactivation proves dissocation to participate in the process of deactivation, while the effects for LDH are explicable on the basis of reversible denaturation alone. Nucleic Acids Res, 1975 Feb, 2(2), 211 - 21 Study of the role of the acceptor stem in the interactions between tRNAs and aminoacyl-tRNA synthetases; Bonnet J et al.; Several studies have clearly demonstrated that the end of the acceptor stem was a very important area determining the aminoacylation properties of tRNAs . However the attempts to measure the contribution of this region to the binding of tRNAs to aminoacyl-tRNA synthetases have led to contradictory results . We report here the stepwise degradation of yeast tRNA-Phe and tRNA-Val from their 3' terminus, up to the seventh nucleotide : the affinity of each of the degraded-tRNA for their cognate aminoacyl-tRNA synthetase was compared to that of intact tRNA and it was found that these affinities are not significantly decreased when compared to those of the intact tRNAs. J Antibiot (Tokyo), 1975 Feb, 28(2), 132 - 5 Stability studies with amphotericin B and amphotericin B methyl ester; Bonner DP et al.; In solid form, amphotericin B and amphotericin B methyl ester free base exhibit similar stability . Acid salts of the methyl ester derivative stored under identical conditions are less stable . In solution, amphotericin B is generally more stable than its methy ester salts . However, when pH is adjusted to 6.0 and storage temperature held at 5 degrees C the methyl ester salts reflect the stability exhibited by the parent compound, amphotericin B. Biochem J, 1975 Feb, 146(2), 409 - 16 Membrane-lipid unsaturation and mitochondrial function in Saacharomyces cerevisiae; Watson K et al.; The lipid composition of yeast cells was manipulated by the use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae . There was a 2-3-fold decrease in the concentration of cytochromes a+a3 when the unsaturated fatty acid content of the cells was decreased from 60-70% of the total fatty acid to 20-30% . The amounts of cytochromes b and c were also decreased under these conditions, but to a lesser extent . Further lipid depletion, to proportions of less than 20% unsaturated fatty acid, led to a dramatic decrease in the content of all cytochromes, particularly cytochromes a+a3 . The ATPase (adenosine triphosphatase), succinate oxidase and NADH oxidase activities of the isolated mitochondria also varied with the degree of unsaturation of the membrane lipids . The lower the percentage of unsaturated fatty acid, the lower was the enzymic activity . Inhibition of mitochondrial ATPase by oligomycin, on the other hand, was not markedly influenced by the membrane-lipid unsaturation . Npn-linear Arrenius plots of mitochondrial membrane-bound enzymes showed transition temperatures that were dependent on the degree of membrane-lipid unsaturation . The greater the degree of lipid unsaturation, the lower was the transition temperature . It was concluded that the degree of unsaturation of the membrane lipids plays an important role in determining the properties of mitochondrial membrane-bound enzymes. Biochemistry, 1975 Jan 28, 14(2), 362 - 8 Relationship between fluorescence and conformation of epsilonNAD+ bound to dehydrogenases; Luisi PL et al.; This work reports on the interaction of the fluorescent nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+) with horse liver alcohol dehydrogenase, octopine dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase from different sources (yeast, lobster muscle, and rabbit muscle) . The coenzyme fluorescence is enhanced by a factor of 10-13 in all systems investigated . It is shown that this enhancement cannot be due to changes in the polarity of the environment upon binding, and that it must be rather ascribed to structural properties of the bound coenzyme . Although dynamic factors could also be important for inducing changes in the quantum yield of epsilonNAD+ fluorescence, the close similarity of the fluorescence enhancement factor in all cases investigated indicates that the conformation of bound coenzyme is rather invariant in the different enzyme systems and overwhelmingly shifted toward an open form . Dissociation constants for epsilonNAD+-dehydrogenases complexes can be determined by monitoring the coenzyme fluorescence enhancement or the protein fluorescence quenching . In the case of yeast glyceraldehyde-3-phosphate dehydrogenase at pH 7.0 and t = 20 degrees the binding plots obtained by the two methods are coincident, and show no cooperativity . The affinity of epsilonNAD+ is generally lower than that of NAD+, although epsilonNAD+ maintains most of the binding characteristics of NAD+ . For example, it forms a tight complex with horse liver alcohol dehydrogenase and pyrazole, and with octopine dehydrogenase saturated by L-arginine and pyruvate . One major difference in the binding behavior of NAD+ and epsilonNAD+ seems to be present in the muscle glyceraldehyde-3-phosphate dehydrogenase . In fact, no difference was found for epsilon NAD+ between the affinities of the third and fourth binding sites . The results and implications of this work are compared with those obtained recently by other authors. Eur J Biochem, 1975 Jan 15, 50(3), 475 - 81 The conformation of adenosine diphosphoribose and 8-bromoadenosine diphosphoribose when bound to liver alcohol dehydrogenase; Abdallah MA et al.; 8-Bromo-adenosine diphosphoribose (br8 ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr8AD+) which are analogues of the coenzyme NAD+, were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods . Nbr8AD+ is active in alcohol dehydrogenase complexes studied by crystallographic methods . Nbr8AD+ is active in hydrogen transport and br8ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase . X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution . The conformations of these analogues were determined from the X-ray data . It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD+, when NAD+ is bound to lactate and malate dehydrogenase . br8ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD+, in contrast to the syn conformation found in 8-bromo-adenosine . The overcrowding at the 8-position is relieved in br8ADP-Rib by having the ribose in the 2' endo condormation instead of the usual 3' endo as in ADP-Rib and NAD+. Biochim Biophys Acta, 1975 Jan 14, 375(1), 69 - 86 Magnetic resonance studies on the mitochondrial divalent cation carrier; Case GD; Measurements of water proton spin relaxation enhancements (epsilon) can be used to discriminate high-affinity binding of Mn-2+ or Gd-3+ to biological membranes, from low-affinity binding . In rat liver mitochondria, epsilon b values of approx . 11 are observed upon binding of Mn-2+ to the inner membrane, while internal or low-affinity binding remains invisible to this technique . Energy-driven Mn-2+ uptake by liver mitochondria results in the subsequent decay of epsilon . Comparison of epsilon with the initial velocity of Mn-2+ uptake in rat liver mitochondria reveals a linear correlation, which holds at all temperatures between 0 degrees C and 40 degrees C, regardless of the mitochondrial protein concentration . Consequently, enhancement appears to reflect the binding of Mn-2+ to the divalent cation pump . Binding of Mn-2+ to blowfly flight muscle also results in substantial epsilon, which is associated with the glycerol-1-phosphate dehydrogenase instead of divalent cation transport . Consequently, no decay in epsilon due to uptake occurs after Mn-2+ is bound . Lanthanide ions are also bound and transported by mitochondria . Addition of Gd-3+ to pigeon heart or rat liver mitochondria results in epsilon b approximately equal to 5-6, which decays with similar kinetics in both systems . The uptake velocity of Gd-3+ in rat liver mitochondria is about 1/6 the rate with which Mn-2+ is transported . Lanthanides also diminish epsilon due to the addition of Mn-2+, and greatly retard the Mn-2+ uptake kinetics . The presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone depresses epsilon upon addition of Mn-2+ or Gd-3+ and also uncouples energy-driven uptake . On the other hand, prolonged anaerobic incubation in the presence of antimycin and rotenone exhausts the mitochondria of their energy stores, blocks the uptake of Mn-2+, but does not affect epsilon significantly . Evidently, the uncoupler-induced disappearance of divalent cation binding sites is not the result of "de-energization" . Measurements of epsilon at several NMR frequencies indicate a correlation time (tau b) for carrier-bound Mn-2+ in rat liver mitochondria between 20 ns and 4 ns as one varies the temperature between 10 degrees C and 30 degrees C . The 13 Kcal/mole activation energy for tau b suggests that the 11 ns time constant at room temperature represents the movement of the Mn-11-carrier comples . On the other hand, tau b is probably approx . 100 times too short to represent the rotational motion of a carrier protein . Apparently, Mn-2+ binds to a small arm of the carrier which moves independent Biochim Biophys Acta, 1975 Jan 6, 378(1), 125 - 32 An acridine probe into the physiological state of the cell; Ito T et al.; Acridine orange was used as a probe to look into the physiological state of the yeast cell, particularly as regards the change in the properties of the membrane (which acts as a barrier against the incoming acridine orange) and the availability of binding sites for acridine orange in chromosomal DNA during growth . After acridine orange had been introduced into the cell, the genetic change at a specific locus with incubation time was measured photodynamically . A three-fold increase in the rate of penetration of acridine orange into the cell was observed, for instance, in going from the resting phase to the dividing phase . A five-fold increase was observed in the number of binding sites in chromosomal DNA under the same transition of the cell . These two parameters may be useful as a measure of the physiological changes in the cell . Some environmental factors such as pH and temperature were also demonstrated to affect the parameters. Acta Biochim Biophys Acad Sci Hung, 1975, 10(3), 171 - 6 Inhibition by quinaldate of dehydrogenases; Konig T et al.; Quinaldate (quinoline-2-carboxylate) inhibits pyridine-, and flavin nucleotide-dependent dehydrogenases, both inside the mitochondria and in isolated form . Other mitochondrial functions and some other isolated enzymes (with one exception) are not influenced by quinaldate at all . Thus, quinaldate can be regarded as a specific "dehydrogenase inhibitor" . The inhibition of alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) by quinaldate is of mixed type, both with respect to NAD+ and ethanol or lactate, respectively . The Ki for alcohol dehydrogenase is 4.5 mM, that of lactate dehydrogenase 7.5 mM . It can be assumed that the inhibition by quinaldate of dehydrogenases is a consequence of its binding to that part of the active centre which takes part in the dehydrogenation itself and might possess very similar structure in all dehydrogenases. J Surg Oncol, 1975, 7(5), 337 - 45 Offsetting toxicity of antineoplastic agents; Cook ES et al.; PCO, a yeast extract, offsets at least in part the mitotic inhibitory effect of methotrexate and fluorouracil on bone marrow cells in vitro but increases the antimitotic activity of the drugs on ascites Krebs-2 carcinoma under similar conditions . In vivo, PCO enhances the action of methotrexate against the L-1210 lymphoid leukemia and does not interfere with the effectiveness of fluorouracil against the ascites Krebs-2 tumor. Physiol Chem Phys, 1975, 7(2), 167 - 75 The effect of sodium bisulfite on the melting profiles of nucleic acids and on their respective nucleosides; Marfey P et al.; The effect of sodium bisulfite (0.27 M, pH 7) on melting behavior of DNA, yeast RNA and their respective nucleosides was studied . It was found that bisulfite added not only to pyrimidine bases but also to purine bases of nucleic acids and of nucleosides . The addition products were stable at higher temperatures but reverted to parent compounds at room temperature . The only exception was the addition product of uridine which was stable at room temperature and could be isolated by paper chromatography in a 42-62% yield . Heating of DNA solutions in the presence of bisulfite to 95 degrees C caused a 90% loss of absorbance at 260 nm . On cooling, the absorbance was essentially recovered . When compared to the melting behavior of DNA in 0.27 M NaCl or 0.09 M Na2SO4 (same ionic strength), it was found that bisulfite destabilized double helical structure of DNA and that reversible addition of bisulfite did occur much below the melting temperature of DNA observed in the other two solvents. Scand J Immunol, 1975, 4(2), 145 - 50 Serum factors causing impaired macrophage function in systemic lupus erythematosus; Svensson BO; Leukocytes from patients with systemic lupus erythematosus (SLE) and healthy controls were cultured for 4 days in either SLE serum or control serum . The remaining monolayer of monocyte-derived macrophages was allowed to ingest yeast cells . Macrophages (from SLE patients or controls) incubated in SLE sera presented impaired phagocytic activity and glass adherence compared with cells grown in control sera . SLE sera contain one or more factors that impair macrophage function; IgG-containing immune complexes may be one such factor. Radiat Environ Biophys, 1975, 11(4), 319 - 25 The repair of potentially lethal damage; an alternative approach; Chadwick KH et al.; An alternative approach to the explanation and analysis of post-irradiation effects is presented . The analysis is based on the repair of a radiation-induced lesion and it is shown that the experimental results are compatible with the repair of one type of critical lesion involved in cell reproductive death . The nature of the lesion with respect to potentially lethal damage and sub-lethal damage is discussed. J Biochem (Tokyo), 1975 Jan 1, 77(1?), 81 - 8 Kinetic studies of carboxypeptidase Y . II . Effects of substrate and product analogs on peptidase and esterase activities; Bai Y et al.; Reversible inhibition of the peptidase and esterase activities of CPase Y {EC 3.4.12.1} was investigated with substrate and product analogs known to be inhibitors or effectors of pancreatic carboxypeptidases A or B {EC 3.4.12.2 or 3} . L-Amino acids and NH2-blocked L-Amino acids showed competitive-type inhibition, whereas their D-enantiomers caused less inhibition than the L-enantiomers, and showed non-competitive or mixed-type inhibition . Some phenylalanine analogs, e.g . beta-phenylpropionic acid and t-cinnamic acid, were also reversible inhibitors of CPase Y . The type of these inhibitors and their K1 values were generally parallel for both the peptidase and esterase activities. J Biochem (Tokyo), 1975 Jan 1, 77(1?), 69 - 79 Kinetic studies of carboxypeptidase Y . I . Kinetic parameters for the hydrolysis of synthetic substrates; Hayashi R et al.; Kinetic parameters for carboxypeptidase Y {EC 3.4.12.1}, characterized as a nonspecific enzyme, are given for the hydrolysis of a series of acylated peptides, acylated amino acid esters, and amides . We confirmed that the enzyme released COOH-terminal proline and beta-alanine at an appreciable rate, as well as neutral amino acids with aromatic and aliphatic side chains at a very high speed . The rates of hydrolysis of ester and amide substrates were compatible with those produced by chymotrypsin {EC 3.4.21.1} . Stereospecificity was also demonstrated by the failure to hydrolyze peptide, ester, amide, and anilide substrates containing a D-amino acid . The effects of pH, solvents, and salt concentrations on the kinetic parameters of hydrolysis of peptide and ester substrates are also described. Proc Natl Acad Sci U S A, 1975 Jan, 72(1), 172 - 6 Biosynthesis of polypeptides of cytochrome c oxidase by isolated mitochondria; Poyton RO et al.; Yeast mitochondria, incubated with radioactive amino acids in a "protein-synthesizing mixture" containing an oxidizable substrate and an ATP regenerating system, have been shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to incorporate label into polypeptides equivalent in molecular weight and relative amount ot those made in vivo in the presence of cycloheximide . The ability of these isolated mitochondria to synthesize "native" polypeptides was assessed by examining the incorporation of label into subunits of cytochrome c oxidase (EC 1.9.3.1) . An analysis of immunoprecipitates formed by incubating cholate extracts of labeled mitochondria with an antiserum against holocytochrome c oxidase revealed that label was incorporated into three polypeptides of sizes equivalent to those of cytochrome c oxidase subunits I, II, and III, shown from earlier studies in vivo to be translated on mitochondrial ribosomes . Further evidence that these polypeptides made in vitro are "native" and identical to subunits I, II, and III was provided by the observation that labeled polypeptides equivalent in size to subunits I-III- ARE ALSO IMMUNO-PRECIPITATED BY ANTISERUM AGAINST SUBUNITS V plus VII, an antiserum that can precipitate subunits I, II, and III only when they are complexed to the cytoplasmically synthesized subunits, V and VII, of the enzyme . These results suggest that isolated mitochondria are capable of synthesizing three subunits of cytochrome c oxidase and assembling them into a holoenzyme. C R Seances Soc Biol Fil, 1975, 169(3 Suppl), 759 - 65 {Radiobiological studies on the behavior of normal and tumorous plant tissues cultivated in vitro}; Jonard R et al.; Vegetable tumoral tissue are more radiosensitive than the homologous normal tissue . Tumoral tissue are more stimulated by yeast extract and yeast s-ARN than normal tissue . Yeast extracts and s-RNA extracts from yeast also restore more the growth of tumorous tissue than normal tissue, which were subjected to gamma radiation from 60Co . A mixture of mononucleotides obtained from fractionated s-RNA has the same stimulatory effect . It is suggested that the active principle from yeast and s-RNA is a cytokinin like substance, which have been detected in yeast extract and in soluble ribonucleic acid from yeast. Z Naturforsch {C}, 1975 Jan-Feb, 30(1), 117 - 9 Influence of spermine on some membrane-disturbing actions; Elferink JG; The influence of spermine on the potassium loss from yeast cells as a consequence of membrane-disturbing actions was investigated . Spermine interfered strongly in the action membrane-active bactericides on yeast cells; this interference resembles the action of certain metal ions . Spermine provides a protection against the positively charged parasosaniline and accomplishes a strong potentiation of the action of the negatively charged sodium dodecyl sulphate. J Lipid Res, 1973 Nov, 14(6), 625 - 31 Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A; Hulcher FH et al.; A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase {acylating CoA}, EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate . Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) . Sodium arsenite forms a complex with the dithiol, but not with monothiols . Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly . The absorbance due to the coenzyme A-5,5'-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent . After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from epsilon(max) = 1.36 x 10(4) at 412 nm . The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical . This method provides an immediate assay . Recovery of reduced coenzyme A was 98.7% . The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A . It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction . The apparent K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 10(-4) M . This method was compared with the dual-label method at high and low levels of activity . The data were not statistically different.
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