[Construction of the gene library of symbiotic nitrogen-fixing Rhizobium lupini]. / Poluchenie biblioteki genov simbioticheskoi azotfiksiruiushchei bakterii Rhizobium lupini.

The gene bank of the symbiotic nitrogen-fixing bacterium Rhizobium lupini (effective strain 359a) was constructed on plasmid pAYC31 that was used to transform Escherichia coli C6000. The bank contains 6600 clones. Restriction analysis showed that the size of the mean insertion fragment in the plasmid in 6.5 kb.

[Enzymes of thiol-disulfide metabolism of proteins]. / Fermenty tiol-disul'fidnogo obmena belkov.

Occurrence, properties and physiological role of protein disulfide reductases (EC 1.6.4.4 and 1.8.4.2), protein disulfide isomerase (EC 5.3.4.1), and thiol oxidase (EC 1.8.3.2) catalyzing thiol-disulfide interchange reactions in proteins are reviewed with a particular emphasis on seed storage proteins. An important role of the enzymes in the formation and degradation of seed storage protein complexes is discussed.

[Structural role of histidine residues in NAD(P)-glutamate dehydrogenase from the bovine liver]. / Strukturnaia rol' ostatkov gistidina NAD(P)-glutamatdegidrogenazy pecheni byka.

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.

[Kinetic characteristics of glutamine synthetase from the chloroplasts of pea leaves]. / Kineticheskie kharakteristiki glutaminsintetazy Khloroplastov list'ev gorokha.

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.

[Mechanism of inhibition of pea chloroplast glutamine synthetase by methionine sulfoximine]. / Mekhanizm ingibirovaniia glutaminsintetazy khloroplastov gorokha metioninsul'foksiminom.

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.

[Functional groups involved in the nitrate reductase activity of milk xanthine oxidase]. / Issledovanie funktsional'nykh grupp, uchastvuiushchikh v nitratreduktaznoi aktivnosti ksantinoksidazy moloka.

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.

[Secondary structure and functional groups of the active center of glutamine synthetase of pea chloroplasts]. / Vtorichnaia struktura i funktsional'nye gruppy aktivnogo tsentra glutaminsintetazy khloroplastov gorokha.

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.

[Properties of metlegoglobin reductase from lupin root nodules]. / Svoistva metlegoglobinreduktazy kluben'kov liupina.

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.

[Two forms of glutamine synthetase from pumpkin leaves]. / Dve formy glutaminsintetazy v list'iakh tykvy.

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.

[Properties of metlegoglobin reductase from lupine nodules]. / Metlegoglobinreduktaza kluben'kov liupina. Hekotorye svo¿istva.

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.

[Nitrate and nitrite reductase activity of milk xanthine oxidase]. / Nitrat-i nitritreduktaznaia aktivnosti ksantinoksidazy moloka.

The nitrate and nitrite reductase activities of milk xanthine oxidases were studied. The optimal conditions for manifestation of these activities were found. A possible mechanism of electron transport to nitrate and nitrite inside the enzyme is discussed.

Isolation of cofactor common to molybdenum-containing enzymes: nitrate reductase from lupine bacteroids and xanthine oxidase from milk.

A quantitative method for the anaerobic isolation of a molybdenum cofactor from two molybdenum-containing enzymes, nitrate reductase from the bacteroids of lupine nodules and xanthine oxidase from milk, is described. It was established that the cofactor consists of an aromatic component and a number of amino acid residues bound to it. The structural and catalytic function of the molybdenum cofactor in the enzyme was established.

[Metlegoglobin reductase from lupine nodules. Purification and properties]. / Metlegoglobinreduktaza kluben'kov liupina. Ochistka Svoistva.

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.

[Ferredoxin-dependent glutamate synthase of Chlorella]. / O ferredoksin-zavisimoi glutamatsintaze khlorelly.

Ferredoxin-dependent glutamate synthase has been found in cells of thermophylic Chlorella strain Ch. pyrenoidosa 82T. The enzyme is active with its own ferredoxin and that of Spirulina. Glutamate synthase activity increases during nitrogen starvation and than decreases in the course of successive ammonium assimilation. The scheme of ammonium assimilation in Chlorella pyrenoidosa 82T cells is proposed.