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Biomedical subjects

V L Kretovich

Publications and source records attributed to V L Kretovich.

At least 19 recordsLinked to original sources
PubMed1985

[Structural role of histidine residues in NAD(P)-glutamate dehydrogenase from the bovine liver].

Photooxidation of bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3) in the presence of methylene blue at a low light intensity occurs in two stages. At the first stage, the duration of which depends on temperature and dye concentration, a slight activation is observed simultaneously with the oxidation of two histidine residues. At the second stage, the inactivation is concomitant with the oxidation of three histidine and one tryptophan residues. The inactivation is a first order reaction (k = 3,22 X 10(-2) min-1) and is correlated with changes in the circular dichroism spectra. These data testify to the structural role of histidine residues in the GDH molecule. The kinetic behaviour of GDH during its modification with diethylpyrocarbonate (DEP) depends on pH and the reagent concentration. Four histidine residues undergo carbethoxylation at pH 6.0 and 7.5, but the modification rate is much higher at pH 7.5. At low DEP concentrations, a remarkable activation is observed with a simultaneous modification of one histidine residue, which is independent of pH. At high DEP concentrations, a rapid inactivation takes place at pH 7.5. Treatment of the carbethoxylated inactive enzyme with hydroxylamine results in the deacylation of histidine residues without any noticeable reactivation. The data on the combined effect of DEP and pyridoxal-5'-phosphate suggest that GDH inactivation by DEP at pH 7.5 is a result of modification of an essential epsilon-NH2 group of lysine-126.

Animals↗
PubMed1983

[Secondary structure and functional groups of the active center of glutamine synthetase of pea chloroplasts].

The circular dichroism spectra of glutamine synthetase (EC 6.3.1.2) from pea chloroplasts were recorded. Based on these spectra the percentage of alpha-helix sites, beta-structures, beta-bends and disordered sites of the polypeptide chain was calculated and was found equal to 23, 57, 1 and 23%, respectively. Data from protein photooxidation in the presence of methylene blue and the type of pH-dependence of pKm suggest that glutamate binding takes place on the imidazole ring of the histidine molecule. The inhibition of native glutamine synthetase by p-chloromercurybenzoate and the presence of free SH-groups in the enzyme molecule (approximately two SH-groups per monomer) suggest that these groups are the functional groups of the enzyme active center.

Binding Sites↗
PubMed1983

[Mechanism of inhibition of pea chloroplast glutamine synthetase by methionine sulfoximine].

In the presence of ATP and Mg2+ L-methionine sulfoximine irreversibly inhibits homogeneous glutamine synthetase (EC 6.3.1.2) from pea chloroplasts (I0.5 = 1.0 x 10(-7) M; Ki = 6.25 . 10(-8) M. Glutamate (but not NH4Cl) exerts a protective effect, which is enhanced when glutamate and NH4Cl are simultaneously present in the reaction mixture. The inhibiting action of L-methionine sulfoximine with respect to glutamate is of a mixed type. ATP and Mg-ATP produce the same non-competitive protective effect on L-methionine sulfoximine. The data obtained suggest that the formation of a quaternary complex (or a transition state) between the enzyme and all its substrates is essential for the catalysis.

Chloroplasts↗
PubMed1983

[Kinetic characteristics of glutamine synthetase from the chloroplasts of pea leaves].

The kinetic properties of glutamine synthetase (EC 6.3.1.2) isolated from pea chloroplasts and purified according to the previously developed procedure have been investigated. The pH optimum for the enzymatic reaction in the presence of Mg2+ and Mn2+ are 7.5-7.6 and 5.5, respectively. The corresponding values of the activation energy per enzyme monomer (Mr = 60 000) are equal to 2900 and 1190 cal/mole. With Mg2+ the values of Km(app.) for NH4+, NH2OH, L-glutamate (+NH4+), L-glutamate (+NH2OH), ATP(+NH4+ and NH2OH) and Mg-ATP (+NH4+ and NH2OH) are 0.64, 17.5, 5.6, 7.0, 1.3 and 0.74 mM, respectively.

Cations↗
PubMed1983

[Functional groups involved in the nitrate reductase activity of milk xanthine oxidase].

Milk xanthine oxidase possesses the nitrate reductase activity at pH 5.2; the pH optimum of the xanthine oxidase activity for the enzyme lies at 9.6. After removal of FAD and binding of Mo and Fe with a simultaneous measurement at the pH optima of the above activities it was found that only the Mo-containing site is necessary for the nitrate reductase activity. The switch-over of the enzyme from the xanthine oxidase to the nitrate reductase activity is associated with considerable conformational changes of the enzyme molecule.

Animals↗
PubMed1982

[Properties of metlegoglobin reductase from lupin root nodules].

The interaction between metlegoglobin reductase from lupin root nodules cytosol and some substrates and inhibitors was studied. The Km values for electron acceptors: dichlorophenol indophenol, potassium ferricyanide, methylene blue and cytochrome c were 5.7 x 10(-5), 2.1 x 10(-5), 1.75 x 10(-4) and 2.5 x 10(-5) M, respectively. The Km value for electron donor NADH was 2.4 x 10(-5) M. Hydroxymercurybenzoate and ethylmaleimide inhibited the metlegoglobin reductase activity; the enzyme activity was also inhibited by NAD. Metlegoglobin reductase was inhibited by quinacrine, which confirmed the flavoproteid nature of the enzyme earlier discovered by the authors. Cytochrome b5 from rabbit liver microsomes can be an electron intermediate during cytochrome c reduction by metlegoglobin reductase. The temperature optimum of metlegoglobin reductase is 40 degrees. The enzyme is comparatively thermostable; and was inactivated by 85% only after 5 min heating at 100 degrees.

Animals↗
PubMed1981

[Two forms of glutamine synthetase from pumpkin leaves].

Two molecular forms of glutamine synthetase localized in the cytoplasm and in chloroplasts, respectively, were detected in pumpkin leaves. Ammonium infiltrated into intact pumpkin leaves activated the synthesis of both enzyme forms. Glutamine synthetase from chloroplasts and the cytoplasmic enzyme were purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose DE-32, selective adsorption on potassium phosphate gel and preparative electrophoresis in polyacrylamide gel. The molecular weights of both forms of glutamine synthetase as determined by gel-filtration through Sephacryl S-200 are equal to 370,000 and 480,000, respectively. During SDS polyacrylamide gel electrophoresis the enzymes from both sources produced polypeptide chains with respective molecular weights of 50,000 and 58,000. The amino acid composition of the enzymes differed considerably. The content of alpha-helix moities in the chloroplast and cytoplasmic enzyme made up to 17% and 34%, respectively. In the presence of Mg+ the pH optima for the enzymes were equal to 7.75 and 7.25, respectively, and the Km values for L-glutamate were 46 and 13 mM, respectively. It may be concluded that the enzyme forms under study are isoenzymes.

Amino Acids↗
PubMed1980

[Properties of metlegoglobin reductase from lupine nodules].

The purification and properties of metlegoglobin reductase from lupine (Lupinus luteus L.) nodules are described. The purification procedure results in a 1056-fold purification of the enzyme with a total yield of 21%. The enzyme possesses the NADH-diaphorase activity. Metlegoglobin reductase is heterogenous during electrophoresis and isoelectric focusing. Electrophoresis produces two vicinal active bands, while isoelectrofocusing results in four active fractions. The fraction possessing the highest activity has a pI of 4.4. The enzyme is a flavoprotein, in which all flavins are represented by FAD. The molecular weight of the enzyme is 30 000. In some properties metlegoglobin reductase from lupine nodules is similar to methemoglobin reductase from erythrocytes and metmyoglobin reductase from muscles.

Dihydrolipoamide Dehydrogenase↗
PubMed1979

[Metlegoglobin reductase from lupine nodules. Purification and properties].

The purification procedure and properties of metlegoglobin reductase from the soluble fraction of lupine (Lupinus luteus L.) nodules and from the proteins secreted by bacteroids Rhizobium lupini in vitro are described. The properties of both forms of enzyme were found to be similar. A metlegoglobin reductase preparation purified 125-fold with a yield of 21% was obtained. The enzyme is strictly specific to the cofactor (NADH). No substrate specificity was revealed. The enzyme reduces oxidized cytochrome c, Mb+, Lb+, Hb+ and exygen. The pH optimum for the enzyme is 7,4. The enzyme is inhibited by p-chloromercurybenzoate. In some properties the enzyme from lupine nodules is close to methemoglobin reductase from the erythrocytes. It was shown that apart from metlegoglobin reductase, bacteroids secrete some other proteins, which is indicative of a close interrelationship between the bacteroids and the plant in a symbiotic nitrogen-fixing system.

Cations↗
PubMed1978

[Glutamate dehydrogenases of unicellular green algae: effects of nitrate and ammonium in vivo].

The constitution and control by the inorganic nitrogen source of glutamate dehydrogenases of some unicellular green algae have been studied. The Ankistrodesmus braunii and Scenedesmus obliquus cells contain two different glutamate dehydrogenases, one of which is NADP-specific, the other is active with both NAD and NADP. Their synthesis does not depend on the nitrogen source. The activity of NADP-specific glutamate dehydrogenase increases sharply during nitrogen starvation. In Chlorella pyrenoidosa 82 and Ch. ellipsoidea only one constitutive double specific glutamate dehydrogenase is observed. Its activity does not change depending on the nitrogen nutrition conditions. In the cells of the thermophylic Chlorella strain Chlorella sp. K. ammomium induces a de novo synthesis of NADP-specific glutamate dehydrogenase in addition to the constitutive double specific glutamate dehydrogenase. Thus, the algae tested contain constitutive double specific glutamate dehydrogenase. The NADP-specific enzyme is absent in two Chlorella strains, is constitutive in A. braunii and S. obliquus, and is ammonium-inducible in three thermophylic Chlorella strains.

Chlorophyta↗
PubMed1978

[Ribosomal proteins of pea seeds behaving like anions at pH 8.6].

A group of proteins migrating to the anode at pH 8.6 under polyacrylamide gel electrophoresis was revealed in the total protein of non-dissociated KCl-washed pea seed ribosomes. No proteins with an isoelectric point below pH 4.2 Were found. The presence of acidic proteins in 80 S ribosomes is due to the presence of a specific set of relatively acidic proteins in the total protein of large (5 major and 10 minor components) and small (2 major and 4 minor components) subunits. The mostly acidic proteins are located in the large subunit. The acidic proteins of 60S and 40S subunits are represented by the polypeptide chains with molecular weights from 48 000 to 13 000. The acidic proteins are present in the ribosomes studied in considerably less number than the basic proteins, and the former produce a very weak staining under electrophoretic analysis as compared with the latter. The data obtained suggest that 80S ribosomes of higher plants differ from animal ribosomes by a higher content of relatively acidic proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1978

[Presence of SH-groups and histidine in the active site of Chlorella glutamine synthetase].

The presence of two cysteine residues per each six monomers comprising the oligomer of Chlorella glutamine synthetase (E.C.6.3.1.2) is demonstrated using homogenous enzyme preparation. p-Chloromercuribenzoate (p-CMB) is found to inhibit glutamine synthetase activity, the degree of inhibition depending on the inhibitor concentration. The following enzyme reactivation by dithiotreitol (10(-2) M) was observed only when the enzyme was inactivated with 10(-5) M p-CMB under 15 min. preincubation. Preincubation of the enzyme with 10(-4) M p-CMB for 45 min. did not result in its reactivation. Gel filtration of glutamine synthetase treated with 10(-4) M p-CMB has revealed the dissociation of the enzyme into inactive monomers. Incubation of glutamine synthetase with p-CMB at various pH values, incubation after pre-treatment with urea and experiments with HgCl2 indicate the presence of free and masked inside the globula SH-groups in the enzyme molecule. Competitive character of the enzyme inhibition with p-CMB with respect to ATP indicates that SH-groups of the active site participate in the ATP binding, probably, as Mg-ATP or Mn-ATP complexes. Data on the estimation of ionization constant of glutamate-binding group and experiments on the effect of histidine photooxidation on the enzyme activity indicate the presence of histidine residue in the enzyme active site, which participates in glutamate binding.

Adenosine Triphosphate↗