[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.

[Effect of univalent cations on the glutamate dehydrogenase of chlorella]. / Deistvie odnovalentnykh kationov na glutamatdegidrogenazy khlorelly

Effect of univalent cations (Li+, K+, Na+ and Cs+) on the activity and some kinetic properties of the constitutive and the inducible glutamate dehydrogenases (GDH) of Chlorella pyrenoidosa Pringsheim 82T has been studied. All the cations used activate the inducible GDH and produced no such effect on the constitutive GDH. From the analysis of the kinetic behaviour in the presence of K+ the conclusion was made that K+ promotes and stabilyzes a catalitically advantagenous conformation of the inducible GDH. This phenomenon appears to have a physiological meaning, because of a higher K+ concentration in Chlorella cells (about 0.1 M) and its important role in metabolism.

[Effect of p-chloromercuribenzoate on glutamine synthetase in the food yeast Candida tropicalis]. / Vliianie p-khlormerkuribenzoata na glutaminsintetazu kormovykh drozhzhei Candida tropicalis.

The effect of p-CMB on the activity of glutamine synthetase in the fodder yeast Candida tropicalis was studied in the synthetase reaction in Mg-, Mn-, and Co-activated systems and in the transferase reaction. The activity of glutamine synthetase was inhibited by pCMB, the degree of inhibition depending on the presence of bivalent cations of metals. Preliminay incubation of the enzyme with p-CMB stimulated the action of the latter, whereas metals increased the stability of the enzyme to pCMB. If the enzyme preparation was purified, it became more susceptible to the action of p-CMB. The transferase activity was also inhibited by p-CMB but to a less extent that the synthetase reaction.

[Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components]. / Reguliatsiia metabolizma glutamina u Chlorella pyrenoidosa. Reguliatsiia aktivnosti glutaminsintetazy komponentami adenilovoi sistemy.

A decrease of glutamine synthetase (E. C. 6.3.1.2.) activity was observed under the assimilation of ammonium nitrogen in Chlorella. At the same time a decrease of ATP content in Chlorella cells took place. The ATP content was 7-fold decreased, while ADP and AMP contents were 4-fold and 3-fold increased respectively, after 15 min. of Chlorella incubation on "ammonium" medium. Further incubation for 45 min, resulted in gradual increase of ATP content and in decrease of ADP and AMP contents. The value of energy charge in ammonium assimilating Chlorella cells sharply decreased for first 15 min. of incubation and then it normalized gradually. The experiments with glutamine synthetase preparation, isolated from ammonium assimilating cells, have shown that ADP and AMP are strong inhibitors of the enzyme in the presence of Mg2+, and only ADP produces the inhibitory effect in the presence of Mn2+. No enzyme reactivation was observed after the transfer of ammonium assimilating cells into nitrogen-free medium or nitrate medium, the enzyme activity increasing at the expense of enzyme protein synthesis denovo.

[Analysis of ribosomal proteins and ribosomal subunits of pea seeds by electrophoresis in polyacrylamide gel]. / Izuchenie Belkov Ribosom I Ribosomnykh Sub'edinits Semian Gorokha Metodom Zlektroforeza v Poliakrilamidnom gele

The amino acid composition of overall protein of ribosomes and ribosomal subunits of pea seeds has been found typical of ribosomal protein. Electrophoresis in polyacrylamide gel demonstrates that proteins extracted by the solution of 3 M LiCl-4 M urea from purified ribosomes of pea seeds move towards the cathode at pH 2.2 and separate into 41 components. Electrophoresis in a tris-glycine buffer at pH 9.2 does not reveal any substance corresponding to acid proteins. Similar distribution patterns are observed when ribosomal particles are isolated with or without triton (0,5%). The treatment of ribosomes by deoxycholate results in some changes, depending on the detergent concentration. All the protein components detected in ribosomes, except one, are present in the subunits. Proteins of large and small ribosome subunits produced 26 and 21 components respectively in polyacrylamide gel electrophoresis. The distribution patterns of proteins of the two subunits appear to be different. The majority of the components of the large and small subunits differ in mobility. The data obtained suggest considerable specificity of the protein composition of 60S and 40S subunits of 80S ribosomes in higher plants.

[Molecular weight hetergeneity of proteins of the ribosomes and ribosomal subunits from dry pea seeds].

The molecular weight distribution of the total protein of ribosomes and ribosomal subunits isolated from dry pea seeds was studied by electrophoresis in polyacrylamide gel, containing sodium dodecyl sulfate. It was demonstrated that overall protein of 80 S ribosomes is separated into a number of fractions with molecular weights of 10000-64000. Treatment of ribosomes with 0.5 per cent tritone, 0.5 per cent and 1 per cent deoxycholate does not change the general pattern of the molecular weight distribution of ribosomal proteins. The large subunit reveals 19 protein zones (14 major and 5 minor zones), their molecular weights are varying from 10000 to 54000. The majority of proteins of the large subunit have molecular weights of 14000--32000. The molecular weights of 17 protein zones of the small subunit (7 major and 10 minor zones) vary from 10000 to 64000. The majority of proteins of both large and small subunits have molecular weights of 14000--32000. Electrophoretic separation of proteins in the split gel confirmed the fact that the proteins of large subunit differ in molecular weights from those of the small subunit. Thus, ribosomal proteins of pea seeds are shown to produce a typical (for 80S ribosomes) pattern of molecular weight distribution under polyacrylamide gel electrophoresis in the presence of sodium dodecul sulphate.

[Synthesis and breakdown of poly-beta-hydroxybutyric acid in Rhizobium lupini]. / Sintez i raspad poli-beta-oksimaslianoi kisloty u Rhizobium lupini

The effect of various substrates on synthesis and decomposition of poly-beta-hydroxybutyric acid (PHBA) was studied in cell suspensions of the effective strain of Rhizobium lupini and in suspensions of the bacteroids of this strain which were isolated from lupine nodules at different growth stages of the plants. Glucose and beta-hydroxybutyrate were found to be the best substrates for synthesis of PHBA in all variants. The content of PHBA in the presence of these substrates increased in suspensions by 2.0 to 2.5 times during ten hours of incubation. In the presence of succinate, PHBA was actively synthesized only by the bacteroids isolated during the stage of active nitrogen fixation (flowering of the plants). In the absence of exogenous substrates, PHBA was decomposed, especially if ammonium ions were present in suspensions. No synthesis of PHBA was registered in the presence of ammonium and glucose, and the rate of PHBA decomposition was high in this case during the stage of active nitrogen fixation.

[Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids]. / Reguliatsiia metqbolizma glutamina u Chlorella pyernoidosa. Reguliatsiia aktivnosti glutaminsinettazy khlorelly aminokeslotami

Effect of glutamine and its metabolites (amino acids) on Chlorella glutamine synthetase (GS) (E.C.6.3.1.2) in the presence of Mg or Mn was studied. Purified GS preparation was used, isolated from Chlorella grown in the presence of NH as a sole nitrogen source. Glutamate, aspartate, alanine and glycine inhibit GS activity in the presence of both Mg and Mn. Tryptophane and valine (up to 15 mM) activate GS in the presence of Mn. Tryptophane inhibits GS in the system with Mg. Sinergistic inhibition was observed under the combined effect of amino acids on GS in the presence of Mn and aspartate or alanine. The change of GS activity observed is supposed to be due to the inhibitory effect of glutamine and amino acids studied, since the glutamine content is increased (in 2.5 times for 5 min) and that of alanine and dicarbonic amino acids (for the following 15 min) under NH assimilation in Chlorella cells.

[Wheat protein disulfide reductase]. / Issledozanie Proteindisul'fidreduktazy Pshchenitsy

Proteindisulphide reductase is isolated and partially purified from wheat seedlings and some properties of the enzyme are studied: pH optimum is 7.4; temperature optimum - 37 degrees C; Km = 2.6-10(-4)M for the substrate (wheat albumin); Km = 7.5-10(-5) M for coenzyme (NADP-H). The enzyme is specific for NADP-H and is not active in the presence of NAD-H. Maximal activity of proteindisulphide reductase is developed in anaerobic conditions. A technique of the estimation of proteindisulphide reductase activity using wheat albumin as a substrate is worked out. The enzyme activity decreases regularly in the corn ripening and increases under germination. It is accompanied by the respective increase or decrease in the amount of disulphide bonds in gluten protein and by changes of physico-chemical characteristics of gluten. Incubation of gluten with the enzyme preparation affects reological properties of gluten (it becomes weaker) and decreases the gluten viscosity of gluten solution.

[Regulation of glutamine metabolism in Chlorella pyrenoidosa. Mechanisms of regulating the activity of glutamine synthetase during ammonia assimilation]. / Reguliatsiia metabolizma glutamina u Chlorella pyrenoidosa. Issledovanie mekhanizmov reguliatsii aktivnosti glutaminsintetazy v protsesse assimiliatsii ammoniia

Glutamine synthetase (GS) (E.C.6.3.1.2) activity in Chlorella cells decreased when NH4+ was added to nitrogen-free growth medium. This GS inactivation had such a rate, that it could not be due to the repression of enzyme synthesis: the GS activity decreased by 20% within 5 minutes of NH4+ assimilation. Glutamine content in cell increased in 2.5 times for this period. In vitro experiments have shown that glutamine is a strong inhibitor of GS from Chlorella grown in the presence of NO3-, and in a less degree--an inhibitor of GS from cells grown in ammonium-containing medium. The data obtained are negative with respect to possible mechanisms of GS activity regulation via adenylation and ATP-dependent destruction of glutamine synthetase.

[Regulation of fodder yeast Candida tropicalis glutamine synthetase activity by the end products of glutamine metabolism]. / Reguliatsiia aktivnosti glutaminsintetazy kormovykh drozhzhei Candida tropicalis konechnymi produktami obmena glutamina

Effect of different products of glutamine metabolism on the activity of glutamine synthetase in the presence of Mg2+, and Mn2+ and Co2+ as cofactors is studied. All the metabolites studied are found to inhibit the glutamine synthetase activity in the presence of any cation listed. The degree and the character of the inhibition by one or other metabolite depended in a considerable degree on the nature of the cation presented in the reaction mixture (Mg2+, Mn2+ or Co2+). The mechanism of the cumulative effect of retroinhibitors under the change of Mg2+ or Mn2+ in the reaction mixture was the same.

[Thermostability of glutamine synthetase in Candida tropicalis feed yeasts in Mg-, Mn- and Co-activated systems]. / Termostabil'nost' glutaminsintetazy u kormovykh drozhzhei Candida tropicalis v Mg-, Mn- i Co-aktiviruemoi sistemakh.

The thermostability of glutamine synthetase of Candida tropicalis was studied in systems activated by Mg, Mn and Co. The enzyme was found to be very thermolabile. Its thermostability depended on the nature of a bivalent cation used in the reaction mixture. Metals stabilized the enzyme since preincubation with metal cations increased the thermostability of glutamine synthetase. Purification of the enzyme preparation increased the stabilizing action of bivalent cations (Mg2+, Mn2+, Co2+).

[Presence of SH-groups and histidine in the active site of Chlorella glutamine synthetase]. / O nalichii SH-Grupp i gistidina v aktivnom tsentre glutaminsintetazy kholerally.

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.

[Effect of dissociating agents on Chlorella glutamate dehydrogenases]. / Vliianie dissotsiiruiushchikh agentov na glutamatdegidrogenazy khlorelly

Effect of urea beta-mercaptoethanol and guanidine--hydrochloride on two Chlorella glutamate dehydrogenases (GDH's) have been studied. Both GDH's are inactivated irreversibly by 2-3 M guanidine hydrochloride. Urea above 4 M rapidly inactivates only NH+4-induced NADP--GDH. Constitutive NAD(P)--GDH is stable in 8M urea solution at room temperature for a long time. beta-Mercaptoethanol does not effect significantly the stability of constitutive GDH in 8m urea. Urea above 1M being in reaction mixture inhibits constitutive GDH in a competitive manner to L-glutamate and uncompetitively regards to alpha-oxoglutarate. Taking this into account, one may conclude that L-glutamate and alpha-oxoglutarate seems to bind to different groups on the enzyme molecule.

[Inhibitory analysis of the respiration of bacteroids from the nodules of yellow lupine]. / Ingibitornyi analiz dykhaniia bakteroidov iz kluben'kov zheltogo liupina

Oxygen uptake and reduction of C2H2 by bacteroids was found to be inhibited by low concentrations of cyanide and azide. However, oxygen uptake was not completely suppressed even by 10(-3) M KCN. Cyanide-resistant respiration was not inhibited by salicyl-hydroxamic acid, and seemed to be accomplished at the account of autoxidable flavo-proteins. A small light-reversible inhibition of respiration by carbon monoxide was found only in the bacteroids with a high rate of nitrogen fixation. Rotenone, antimycin A, and tenoyltrifluoroacetone inhibited oxygen uptake and methylene reduction. Nitrogen fixation, but not respiration, was inhibited by 2,4-dinitrophenol. An electron-transport chain coupled with phosphorylation is supposed to be built into the membranes of the bacteroids. The activity of peroxidase and cytochrome peroxidase was demonstrated in the bacteroids.

[The effect of nitrate and nitrite on the respiration and cytochrome system of Rhizobium lupini]. / Vliianie nitrata i nitrita na dykhanie i tsitokhromnuiu sistemu Rhizobium lupini

Differential spectrophotometry and oxygen electrode were used to study the effect of nitrate and nitrite on the cytochrome system and respiration of Rhizobium lupini cultivated in the conditions of different aeration, and bacteroids of this strain isolated from lupine nodules. In the anaerobic conditions, nitrate (10(-3) M) accepts electrons from the cytochrome system in suspensions of bacteroids and bacterial cells grown in the microaerophilic conditions (12 muM O2), but does not oxidize cytochromes of the cells cultivated in the aerobic conditions (240 muM 32). Nitrate (10(-3) M) oxidizes actively only the cytochrome system of the cells grown in the microaerophilic conditions. Nitrite inhibits oxygen uptake by suspension of the bacteroids and pure culture of the cells; its necessary concentrations and the mode of action depend on the conditions of cultivation. Nitrite, like cyanide, blocks the terminal step in the cytochrome chain of Rh. lupini. Some properties of the cytochrome system are similar in the bacteroids and the bacterial culture grown in the conditions of oxygen deficiency.