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Regulation of activity of acetohydroxyacid synthetase has been studied. L-Valine, one of the end-products of the combined pathway, is the only amino acid to inhibit strongly the enzyme (Ki = 5 × 10-5 M). Sensitivity to the inhibitor is rapidly lost by heating at 35°. The desensitized enzyme is stable at this temperature. In crude extracts, the enzyme is always partly desensitized, but 100% inhibition by valine is reached when the enzyme is assayed in situ, in benzene permeabilized cells. In addition, valine inhibition is more strictly dependent upon pH than activity itself. Kinetic studies of affinity for the substrate or inhibitor show no cooperative effects and valine inhibition seems truly competitive with pyruvate. Specific feedback inhibition by valine constitutes an additional proof that the acetohydroxyacid synthetase activity characterized hi studies reported in the preceding paper, is operative for valine biosynthesis. [ABSTRACT FROM AUTHOR]

Crude extracts of Candida utilis contain two types of 6-phosphogluconate dehydrogenase. One can be obtained in the crystalline form and has already been described and studied. In the present paper the purification of the second one, together with the differences in the properties between the two purified proteins, is reported. The two types of enzyme have identical pH optimum, same Km for the substrates and same specificity. They differ in amino acid composition, molecular weight, electrophoretic mobility, stability to pH and heat treatment, sensitivity to chlorodinitrobenzene and proteolytic treatment. All evidence indicates a greater instability of the crystalline enzyme in respect to the non-crystalline one and seems to exclude that the two forms of enzyme are an artefact due to the extraction or the purification procedures. [ABSTRACT FROM AUTHOR]

The rates of synthesis and degradation of glucose-6-phosphate dehydrogenase in the liver of male C57BL mice are followed by a modified immunochemical method. Mice are given labeled amino acids for a predetermined period, and the labeled enzyme is isolated from liver homogenates with goat anti-enzyme, followed by rabbit anti-goat IgG serum. The precipitates are either counted directly or analyzed on acrylamide gels. The rate of glucose-6-phosphate dehydrogenase synthesis is found to be independent of the state of induction or repression of the animal. The synthesis of the enzyme constitutes 0.20-0.25% of the synthesis of all soluble liver proteins. This rate is maintained even during periods of most rapid formation or disappearance of enzyme activity. Quantitative immunoprecipitation shows that similar amounts of precipitable antigen are present in induced and non-induced liver homogenates. The results indicate that the large reversible increase in activity of glucose-6-phosphate dehydrogenase, observed when animals are transferred from a high-fat to a fatless diet, does not involve the synthesis of a new enzyme protein, and that mechanisms involving modulation of existing enzyme molecules ought to be considered for the adaptation of this enzyme in mouse liver.

C3 and C4 α-amino acids bearing art --SO2 H, --SO2SH or --SO3H ω-group are oxidatively deaminated by partially purified D-aspartate oxidase preparations obtained from beef, pig or rabbit kidney. From the Ca sulfur-containing amino acids the partially oxidized sulfur radicals (-SO2H, -SO2SH) are split off during the reaction, originating pyruvate. When a fully oxidized sulfur rascal (-SO3H) is present, sulfopyruvic acid is obtained. The C4 sulfur-containing amino acids all give rise to the corresponding sulfur-containing ketoacids. The Km and V values observed with the three different enzyme preparations for ail the compounds under study are reported. The present results and some observations already known suggest that the substitution of a partially or fully oxidized sulfur radical for the ω-carboxyl group does not greatly affect the specificity of enzymes acting on dicarboxylic amino acids. [ABSTRACT FROM AUTHOR]

Purification of acid phosphomonoesterase from Phaseolus mungo seedlings is reported here. Phosphatase homogeneity was evaluated at 85-90% by several methods. The enzyme is a single polypeptide chain of molecular weight 55000 ± 5000 daltons with serine and leucine as the N and C terminal residues, respectively. A saccharide fraction containing 8% of protein weight as reducing sugar and 3% as amino sugars was found. Attempts to dissociate the saccharidic and protein moieties were unsuccessful. It was concluded that a single phosphatase was active toward phosphomonoesters, phosphoanhydride, and β-D-glycosyl 1-phosphate. [ABSTRACT FROM AUTHOR]

Metabolism of phospholipids in post mortem tissues of liver, kidney, spleen, and heart of guinea pigs was studied. The per cent composition of the individual groups of phospholipids in the above tissues in the fresh state and after a certain period of the dying process taking place under the described conditions is shown. Generally, one can state that phosphatidyl choline is the most important component of phospholipids in these tissues, in liver and in heart representing about 70% of the total amount. They are metabolized relatively faster, while sphingomyelin and phosphatidyl serine are metabolized most slowly. On the basis of the lipid phosphorus determination it is possible to conclude that within 24 h about 20-30% of the phospholipids are decomposed. During the following phase of the dying process no further pronounced decrease of phospholipids takes place. After this period the enzyme system splitting phospholipids is practically inactive. During the dying process an apparent increase of inorganic phosphorus can be observed, which indicates the decomposition of other phosphorus compounds in the course of this process. The relative representation of the individual groups of phospholipids in liver mitochondria is different from that of the whole tissue. Liver mitochondria contain a relatively higher amount of phosphatidyl ethanolamine, a lesser amount of phosphatidyl choline, lysophosphatidyl choline being totally absent. [ABSTRACT FROM AUTHOR]

It has been shown that the initial rates, v, of the formation of aminoacyl-tRNA, catalyzed by phenylalanine-tRNA synthetase, do not depend on the concentration of the substrates ATP and phenylalanine in a simple way. In contrast a normal Michaelis-Menten behaviour has been observed for the substrate tRNA. The reciprocal plots of v versus v/[S] for ATP and phenylalanine are curved and only approach linearity at low and high concentrations of the varied substrate. From these limiting slopes two Km and two V values corresponding to low and high concentrations of ATP and phenylalanine have been determined. Since the reciprocal plots for tRNA are linear, only one Km and V value has been obtained for this substrate. In order to show that this kinetic behaviour is an intrinsic property of the enzyme and not an artifact, a new procedure for the purification of the enzyme has been devised and the purity and stability of the preparation carefully checked. In addition the influence of ammonium ions and of spermine on the initial rates has been measured to verify that the non-linearity of the reciprocal plots is not the result of the substrate acting as a base. Inhibition experiments with tRNAox (a modified tRNA obtained from native tRNA by periodate oxidation of the 2':3'-diol in the 3'-terminal ribose to the corresponding dialdehyde) at low and high phenylalanine concentrations have shown that it is also not a consequence of two fundamentally different reaction mechanisms being followed at the two concentration ranges. On the basis of this and other evidence it is proposed that two mutually interacting sites are engaged, of which one or both are operative, depending on the concentration of the substrates. Studies of pyrophosphate inhibition at low tRNA concentration have provided evidence that the aminoacylation of tRNA proceeds via two catalytic steps in which aminoacyl-AMP occurs as an intermediate. At high tRNA concentrations it has been found that this substrate is adding prior to the dissociation of pyrophosphate. [ABSTRACT FROM AUTHOR]

Two pH-dependent spectral forms of myeloperoxidase have been described. Their absorption maxima in the Soret region were at 432 nm and 426 nm for acid and neutral pH, respectively. Chloride exerts an influence on the spectrum of peroxidase, mainly in acidic medium. Dissociation constants for the myeloperoxidase · chloride complex and Km values for chloride in the chlorination reaction catalyzed by myeloperoxidase were determined. It was found that the affinity of chloride ions to the enzyme decreases with an increase of pH. Catalytic activity of myeloperoxidase in the reaction of incorporation of 36Cl into aliphatic amino compounds such as taurine, β-alanine, ethanolamine and diethanolamine was investigated. Primary amino groups were chlorinated to N-mono- or dichloramines, depending on the pH of the medium. The method of determination of 36Cl-labelled chlorocompounds in the presence of excess Na36Cl was described. The role of myeloperoxidase as chlorinating enzyme in the mechanism of the bactericidal effect on bacteria phagocytized by neutrophilic granulocytes is discussed.

Of the dimeric phosphagen kinases (Mr = 80000) studied, lombricine kinase is the only one which contains only one essential thiol group and one binding site for ADP-Mg. These facts suggest a non-identity between the two subunits of this enzyme. The reversible labeling of the essential thiol group of this protein with 2,4-dinitrofluorobenzene, followed by alkylation of the remaining thiol groups, allowed the separation of the two subunits by means of a Sepharose-mercurial column. The Sepharose-mercurial was prepared by treatment of Sepharose 4B with p-aminophenyl mercuric acetate. Amino-acid analyses, N- and C-terminal determinations and fingerprintings were performed to detect the differences in the primary structure between the two subunits. Furthermore, the Sepharose-mercurial column was used to purify the tryptic peptide containing the essential thiol group of lombricine kinase. By this process, it was possible to obtain, in one step, the peptide in a pure state. It is interesting to point out the applicability of this method for the purification of peptides or proteins containing essential thiol groups which can be specifically labelled by reversible inhibitors.

Identifies the three fragments from the A protein cistron of coliphage R17 ribonucleic acid. Amino acid sequence; RNA translation into protein; Enzyme digestion.

A high ribonuclease activity has been detected in bull seminal plasma; two major fractions (RNAase BS-1 and RNAase BS-2) have been identified, which are responsible for such activity and one of the two, RNAase BS-1, has been purified and crystallized. It has a molecular weight of 29000, an isoelectric point at pH 10.3 and A1 cm1% at 278 nm is 4.65. The amino acid composition has been determined, its main features being a high content of basic residues, the absence of tryptophan and cysteine, and the presence of 18 half-cystine residues. The enzyme is produced by the seminal vesicles, and occurs in seminal plasma as a free, soluble component. [ABSTRACT FROM AUTHOR]

Glucarate hydro-lyase, obtained from cells of Pseudomonas A grown on glucarate as the sole source of carbon, was purified 40-fold in 70% yield. The effects of a variety of chemical modifiers and competitive inhibitors on the enzyme activity were studied. Enzyme activity was irreversibly lost by incubating the enzyme with p-chloromercuribenzene sulphonate. (+)-Tartrate, a competitive inhibitor of the enzyme, protected the enzyme from inhibition by this reagent but was not effective when the enzyme was inhibited by coupling it to a diazonium salt. Glucarate, unlike (+)-tartrate, was an effective protecting group against this type of inhibition. The roles of the modified amino acid residues are discussed. [ABSTRACT FROM AUTHOR]

Biosynthetic L-threonine dehydratase was purified from bakers' yeast and crystallized. The enzyme was homogeneous as judged by ultracentrifugation and electrophoresis s20, w was 9.2 S, and the molecular weight was estimated to be approximately 185 000. The enzyme exhibits a slight yellow color. An absorption maximum in the visible region was at 410 nm. Apparent Michaelis constsnt for L-threonine and L-serine are reported. [ABSTRACT FROM AUTHOR]

Both holo- and apo-aspartate-aminotransferase are sensitive to tetranitromethane; however, the inactivation which follows nitration is partial for the holoenzyme, and complete for the apoenzyme. Cysteinyl and tyrosyl residues are modified in both forms of the enzyme; in the apoenzyme a greater number of tyrosines are available to the reagent. The complete inactivation of the apoenzyme is correlated with the loss of only one tyrosyl residue, which is selectively modified under particular conditions. The loss of activity is caused by tile failure of the nitrated apoenzyme to recombine with the coenzyme. [ABSTRACT FROM AUTHOR]

A procedure for the purification of threonine deaminase from Rhodospirillum rubrum is described. The 2200-fold purified enzyme was judged to be pure by disc-gel electrophoresis and ultracentrifugation. Sedimentation velocity centrifugation yielded an δo20,w value of 8.1 S and a D20,w of 4.65 x 10-7 cm2x sec-1. Assuming a partial specific volume of 0.74 g/ml the molecular weight of 164000 was calculated from the Svedberg equation. However, from calibrated Sephadex G-200 columns, a D20,w 4.15x 10-7 cm2x sec-1 was obtained and using this equation, the molecular weight was calculated to be 180000. Electrophoresis of the enzyme on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed that the native enzyme was composed of four subunits, each of about 46,000 molecular weight. In addition to the native tetramaeric molecule, an active octameric species was detected in polyacrylamide gel electrophoresis. The spectrum of the pure enzyme showed absorption maxima at 279 nm and 412nm, characteristic of pyridoxal 5'-phosphate-containing enzymes. Analysis of the cofactor content revealed the presence of 4.2 moles of pyridoxal phosphate per 180000 g of protein. The enzyme displayed a normal Michaelis-Menten substrate saturation curve in the absence of isoleucine. High concentration of isoleucine inhibited the enzyme activity somewhat only at low levels of the substrate; the inhibition was weak as compared to all other biosynthetic threonine deaminases. No other amino acid or any nucleotide tested produced any significant activation or inhibition. In assays at pH values greater than 8.4 a transient build up of a 245 nm absorbing species was observed. [ABSTRACT FROM AUTHOR]

Initial velocity kinetics of the reverse direction showed that the saccharopine dehydrogenasecatalyzed reaction was consistent with a mechanism that the addition and release of reactants to and from the enzyme follow an obligatory order. The product inhibition studies in the forward direction also revealed kinetic patterns consistent with the ordered mechanism where the sequence of addition of substrates being NAD+ and saccharopine, and that of release of products being lysine, α-ketoglutarate and NADH. The various kinetic parameters determined at pH 6.8 and 22° are aisc described. [ABSTRACT FROM AUTHOR]

1. Iodoacetamide was found to inactivate pig heart mitochondrial malate dehydrogenase in a pseudo first order reaction. 2. Two moles of 14C-labelled reagent per mole enzyme are incorporated, into the protein. 3. The reactive residue was identified by amino acid analysis and thin layer chromatography as a histidine alkylated at nitrogen 3. [ABSTRACT FROM AUTHOR]

Thermolysin is a well-known protease which exhibits its specificity against hydrophobic amino acid residues such as L-leucine, L-phenylalanine, etc. whose amino groups donate the susceptible peptide bonds (amino-endopeptidase). The present study was undertaken to investigate the effects of neighboring residues surrounding the sensitive amino acid residues at the amino-side in peptide substrates. For the purpose, a kinetic study was made using various synthetic oligopeptides such as Z-A-(Gly)n-Leu-Ala or Z-Gly-Leu-(Gly)n-B (A or B = various D- or L-amino acid residues; n = 0, 1 and/or 2; the arrow shows the bond split) as substrates. Other kinetic or inhibition studies were also made. These studies indicated that the specificity is affected by at least three amino acid residues on the N-terminal side and by two amino acid residues on C-terminal side from the sensitive amino acid residue (at amino-side) in peptide substrates. The effect of each of the five neighboring amino acid residues for appearance of the specificity was similar with that of the corresponding one which had been observed in a neutral protease of Bacillus subtilis, but that was not completely the same. [ABSTRACT FROM AUTHOR]

Penicilloylated proteins which are found as an impurity in 6-aminopenicillanic acid can be exhaustively digested by pronase to yield amino acids and small peptides. This degradation converts the point polyvalent antigens into a mixture of mostly monovalent haptens which are much less immunogenic and less capable of eliciting immune reactions in sensitized animals. The degradation of the protein impurity can be carded out in 6-aminopenicillanic acid preparations, since even a large excess of 6-aminopenicillanic acid does not appreciably inactivate or inhibit the enzyme. In order to avoid the contamination of 6-aminopenicillanic acid with a proteolytic enzyme, pronase was converted into a water insoluble form by coupling it with bromoacetyl cellulose or carboxymethyl Sephadex. These insoluble derivatives of pronase were found to retain their activity and broad specificity. Thus, they can be readily removed from the medium upon completion of the impurity degradation, to be used repeatedly in a continuous process. [ABSTRACT FROM AUTHOR]

Absorption spectroscopy and circular dichroism measurements have been used to elucidate thc mechanism of lysozyme inactivation on photo-oxidation of the methionyl residues to methionine sulphoxide. The monosulphoxide derivative, which is formed by irradiation of the protein in aqueous solution, displayed spectral properties very similar to those of native lysozyme and retained about 54% enzyme activity. This reduced catalytic efficiency appeared to be due to limited conformational changes of the protein molecule. Striking differences were found, on the contrary, between the optical parameters of native lysozyme and of the di-sulphoxide derivative, which is prepared by irradiation in acetic acid solution and is almost devoid of enzyme activity. Studies on the unirradiated lysozyme demonstrated that this extensive denaturation was caused by the action of the protic solvent used. Whereas the acid-induced denaturation was reversible in the case of unmodified lysozyme, the photo-oxidation of the two methionyl residues irreversibly blocked the molecule in a largely disordered form. These results are discussed with respect to the location of the two methionines in the known atomic model of lysozyme. [ABSTRACT FROM AUTHOR]

The diffusion and sedimentation constants of the pH 6 acetolactate-forming enzyme, determined by analytical ultracentrifugation, were used to calculate a molecular weight of 200000 for the enzyme. The amino acid composition of the enzyme has been worked out, and 3 molecules of cocarboxylase, and 6 atoms of phosphorus were found to be tightly bound to one molecule of the enzyme. In addition, the effect of divalent cations was studied, results indicated that the enzyme has a requirement for manganese. An absolute requirement for cocarboxylase and manganese was not established. Two crystalline forms of the enzyme were studied by electron microscopy using negative staining technique. The plate-shaped crystals were composed of rows of particles measuring approximately 100×60Å. These particles consisted of sub-units, and in some areas a helical organization of the crystal could be seen. The molecular organization was different in the two different crystalline forms of the enzyme. [ABSTRACT FROM AUTHOR]

The leueyl-tRNA synthctase from Bacillus stearothermophilus has been purified 350-fold. The enzyme is stable when stored at 4° in the presence of sulfhydryl protecting compounds. The Km values for leucine and ATP are respectively 240 and 0.84 μM. The ATP-PP1 exchange and the aminoacylation reaction satisfy the Arrhenius law up to 45°. After thermal denaturation the enzyme has been partially renatured. [ABSTRACT FROM AUTHOR]

The effect of acetylation and of sulfhydryl blocking agents on phosphofructokinase from rabbit muscle has been studied. Treatment of phosphofructokinase with N-acetylimidazole led to rapid loss of its allosteric function, as measured by the ability of AMP to stimulate the ATP-inhibited enzyme. The catalytic activity was far less sensitive to acetylation. The loss of allosteric function on acetylation was due to the fact that the modified enzyme was insensitive to ATP inhibition. The presence of ATP during acetylation prevented the loss of allosteric activity. The results indicate that tyrosine residues may play an important role in the inhibitory binding sites of ATP. Incubation of phosphofructokinase with a 50-fold excess of para-chloromercuribenzoate led to complete inactivation of the enzyme, an effect which could be reversed by the addition of excess thiol. Sulfhydryl blocking had no effect on the ability of AMP to stimulate the ATP- inhibited enzyme. The results confirm that sulfhydryl groups are closely associated with the catalytic site of phosphofructokinase, and demonstrate that they are not involved in the inhibition of the enzyme by ATP and its reversal by AMP. [ABSTRACT FROM AUTHOR]

Kallikrein from porcine pancreas is inhibited by heavy metal ions and activated by sulfhydryl compounds or chelating agents. This activation is interpreted as a reversal of heavy metal inhibition. From the experiments the conclusion is drawn that kallikrein is neither a "sulfhydryl protease" nor a metal-dependent protease. The pH dependency of the hydrolysis of α-N-benzoyl-L-arginine ethyl ester by kallikrem at low substrate concentrations indicates three basic groups with pK 7.9 and 5.7 in the free enzyme and pK 6.9 in the enzyme-substrate complex winch participate m catalysis. The pH-dependence resembles trypsin- and especially cholinesterase-catalyzed hydrolyses. From the most alkaline pK value the participation of an amino group and from the other pK values the participation of a histidine residue in kallikrein-catalyzed hydrolysis is inferred. [ABSTRACT FROM AUTHOR]

Aggregates of two and more tRNASer molecules are formed from purified tRNAyeastSer under commonly used storage conditions. The aggregates can also be produced easily by adjusting to pH 3.0 a solution of tRNASer, Ser-tRNASer, or unfractionated tRNA and subsequently adding a neutral Mg++-containing buffer. The various aggregates were characterized by Sephadex elution profiles and by the rates of dissociation at elevated temperatures. At a given temperature, EDTA and phenol increase the rate of dissociation. The tRNASer aggregates were found to accept serine under the conditions of the enzymatic charging of the tRNASer monomer. The rate of charging is lower and the level of charging is only 1/3 to 1/2 of the one, which can be reached with the same aggregate sample after dissociation prior to the assay. Since the control experiments with the aim to explain the serine incorporation as an artefact (enzyme bound radioactivity; aggregate charging via charging of monomeric tRNA) were negative, the idea of a direct charging of the aggregates was accepted. [ABSTRACT FROM AUTHOR]

It is shown that the change in activity and structure of acetyl-δ-chymotrypsin in the pH 7-11 range is not correlated with the ionizations of tyrosine residues. The pKs of the two titratable tyrosines of acetyl-δ-chymotrypsin are found to be 10.2 and 11.5, at 15° (9.6 and 10.8 after correction for electrostatic interactions), and those of the starting acetylated zymogen are 10.5 and 12.1 (10.0 and 11.5 after identical corrections). The plot of log rate of deacetylation of the titratable tyrosines versus pH is linear between pH 9 and pH 12. At a given pH there is a linear relation between the log deacetylation rate and the pKs of the residues, The pH rate profile of acylation of acetyl-δ-chymotrypsin by 4-carboxy-3-nitrophenyl- N,N-diphenylearbamate is bell-shaped, with a maximum at pH 7.9 at 15°, in 0.14 M KCl. The right side of the curve shows a pKapp of 8.9, whether the exposed titrable tyrosines of the enzyme are acetylated or not. A similar PKapp (9.1) is found for the pH-dependent change in optical rotation of the acetylated enzyme, when the exposed tyrosines are acetated. After correction for electrostatic interactions, this apparent pK becomes 8.4 and can be assigned only to the α-aminogroup of the N-terminal isoleucine. [ABSTRACT FROM AUTHOR]

When the lysyl-RNA synthetase from Eseherichia coli was incubated with ATP and L-lysine in the presence of Mg+t, a complex of lysin-RNA synthetase, AMP and lysine was formed, The complex was isolated by filtration on Sephadex G-25. Maximum complex formation was obtained at a molar ratio of substrate to enzyme between 3 and 4, The complex was very stable at low temperatures in the absence of Mg++, while incubation at 45° for 10 minutes gave 75% break- down of the complex. The amino acid of the complex could be transferred to transferRNA in a reversible reaction that did not require Mg++. The transfer reaction was enhanced by the monovalent cations NH4+, Na+, and K+.The 1ysyLRNA synthetase from K. coil recognized about 70% of the lysine specific chams of yeast transferRNA, while the yeast enzyme recogmzed about 80°/a of the lysine specific chains of K. coil transferRNA, The esterification of K. colt transferRXA by the yeast enzyme proceeded at only 20% of the rate obtained with homologous transferRNA, while the E. coil enzyme estenfled yeast transferRNA at about 60% of the rate obtained with E coli transferRNA. [ABSTRACT FROM AUTHOR]

Myeloperoxidase was isolated from leucocytes obtained from the blood of patients suffering from chronic granulocytic leukaemia. The enzyme was purified 850 fold and was homogeneous in ultracentrifuge and free boundary electrophoresis. The molecular weight of the enzyme was found to be about 160,000. The enzyme forms a spectrally characteristic complex with hydrogen peroxide with absorption maximum at 458 mμ. The spectrum of the native enzyme has absorption maxima at 430 and 570 mμ. The reduced enzyme is characterized by a spectrum with absorption maxima at 472 and 637 mμ. The investigated myeloperoxidase catalysed oxidation of amino acids by hydrogen peroxide. Products of the oxidation of amino acids were ammonia, carbon dioxide, and an aldehyde corresponding to the oxidized amino acid. The observed reaction of deamination and decarboxylation is activated by chloride ions. In the presence of the chloride ions the optimum of the reaction is shifted toward the higher pH values. The velocity of the reaction was found to be dependent on the concentration of the amino acid studied. Km values for various amino acids increased in the range 3.4 × 10-4 to 10-3 M in proportion to rising hydrophobic properties of the substrates. Taurine was found to be a competitive inhibitor in the examined reaction, and Ki values were in the range of 2 to 3 × 10-4 M, for different amino acids. [ABSTRACT FROM AUTHOR]

6-Phosphogluconate dehydrogenase has been obtained in the pure, crystalline form from sheep liver by an extensive modification of an earlier purification procedure. The absorption coefficient, A2801%, estimated from disc weight measurement, is 11.4. The amino acid composition is reported. The molecular weight of the enzyme from measurements by several methods is 94000 ± 2000, and there are two subunits in the molecule. From studies of the reaction of the native enzyme with 5,5′-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate, there appear to be two reactive thiol groups per subunit which are essential for activity and are protected by 6- [ABSTRACT FROM AUTHOR]

A study has been performed in order to try to explain some anomalies which are observed in aminoacylation reactions: the extent of aminoacylation often depends upon the experimental conditions, such as the enzyme concentration or the ionic strength. We have demonstrated that these anomalies can be explained, in the case of yeast tRNAIIVal—valyl-tRNA synthetase system, by the existence of two reactions occurring beside the aminoacylation reaction: the spontaneous deacylation of the valyl-tRNAVal and a valyl-tRNA synthetase-catalyzed deacylation of the valyl-tRNAVal. Consequently, any aminoacylation plateau merely reflects the existence of an equilibrium between the aminoacylation reaction and the deacylation reactions. Sometimes the aminoacylation kinetics do not present a plateau but a maximum value followed by a decrease with time. This can be explained by an additional reaction: a valyl-tRNA synthetase-catalyzed degradation of ATP into AMP and PPi in the presence of tRNAVal and of valine. This reaction leads to an accumulation of AMP and PPi favouring the reverse reaction of the aminoacylation. The kinetic constants of these reactions have been determined. Knowing these constants, it is possible to calculate theoretical aminoacylation plateau values which are in good agreement with the experimental ones. These interpretations can probably be extended to all the tRNA aminoacylation systems. [ABSTRACT FROM AUTHOR]

h Lactate dehydrogenase from Streptococcus cremoris US3, which has a molecular weight in solution of 140000, was found by dodecylsulphate-gel electrophoresis to have a minimum molecular weight of 37000. It is, therefore, a tetramer. 2. Approximately 40 tryptic peptides were obtained by fingerprinting and in conjunction with the amino acid composition this indicates that the subunits are similar and probably identical. 3. There is one thiol group per subunit. 4. p-Chloromercuribenzoate inhibits the enzyme. [ABSTRACT FROM AUTHOR]

Previous work on the partial digestion of the native and denatured forms of tRNASer and tRNAPhe from yeast with T1-RNAase was extended. Detailed studies with pancreatic and T2RNAase and a few experiments with sheep kidney nuclease and acid RNAase from hog spleen revealed characteristically different fragmentation patterns for the native and denatured forms of the tRNAs. Apparently it is mainly the miniloop and dihydrouridine regions which are involved in the conformationaI changes responsible for denaturation. The rate constants, activation enthalpies, and activation entropies of the renaturation process were calculated from amino acid acceptance data. When the changes in hyperchromicity at 260 nm of the denatured tRNAs were followed as a function of time, a process was detected which in tRNAPhe is 5-10 times and in tRNASer 10-20 times faster than the renaturation as measured by acceptor activity. The whole net change in absorption, possibly representing the total net change in base stacking, occurs during the fast process. The activation enthalpies and activation entropies are different for the fast and the over-all processes. [ABSTRACT FROM AUTHOR]

The results show that enzyme I of gramicidin S synthetase activates D-leucine in a manner analogous to that of L-leucine (ATP + D-leucine ↔ D-leucyl-adenylate +. PPi) and binds two moles of D-leucine in thioester linkage. It has therefore two thiol sites for D-leucine, compared to one for the L-isomer. One of the sites seems to be the thiol site for L-leucine which is therefore stereo-unspecific. The results also indicate that the site for the formation of the aminoacyl adenylates is the same for the two isomers. In contrast to thioester-bound L-leucine, enzyme-bound D-leucine does not catalyze the ATP-[14C]AMP exchange reaction. At a molar ratio of L-leucine: D-leucine equal to 4, gramicidin S synthesis is almost completely d and the growth of the peptide chain stops at the tetrapeptide stage (D-Phe-L-ProL-Val-L-Orn). Enzyme-bound D-leucine is thus not able to replace L-leucine in the formation of the pentapeptide. [ABSTRACT FROM AUTHOR]

A method is described for enthalpy titration of proteins, using a flow microcalorimeter in combination with a device for continuous pH measurements. The calorimetric titration of lysozyme between pH 1.5 and 12 has been carried out as well as the corresponding potentiometric titration. Enthalpy values for the titrated groups are estimated and discussed in comparison with reported data for free amino acids. [ABSTRACT FROM AUTHOR]

The amino acid composition of octopine dehydrogenase from muscles of Pecten maximus was determined. The calculated partial specific volume of 0.74 allowed us to evaluate the molecular weight of the enzyme as 38000. It is concluded that this enzyme consists of a single polypeptide chain on the basis of the molecular weight determined from equilibrium ultracentrifugation in 6 M guanidine hydrochloride and from electrophoresis on polyacrylamide gel containing sodium dodecylsulfate as well as on the basis of the number of peptides obtained by tryptic digestion. The presence of one essential -SH group per mole of enzyme and of one binding site for NADH are also consistent with the existence of a single active center. No NH2-terminal amino acid can be detected by reaction with dansyl chloride and with phenylisothiocyanate. Controlled carboxypeptidase action showed that the C-terminal amino acid is lysine. [ABSTRACT FROM AUTHOR]

Determination of the molecular weight of valyl and isoleucyl tRNA synthetases under denaturing conditions leads to the conclusion that both enzymes are made ora single polypeptide chain of a molecular weight of 110000 approximately. The presence of one amino acid binding site per chain was determined by equilibrium dialysis. [ABSTRACT FROM AUTHOR]

The rate of decomposition of tetranitromethane in the presence of enzyme-substrate complexes of L-aspartate: 2-oxoglutarate aminotransferase has been studied; the intermediate Skiff base of aspartate aminotransferase and erythro-β-hydroxy-L-aspartate was shown to undergo nitration. The catalytic course of nitration of the complex is indicative of predominant modification of the substrate part of the carbanion. The presence of carbanion was demonstrated in the course of both model and enzymatic transamination. [ABSTRACT FROM AUTHOR]