Search PubMed⌕ Search

Biomedical subjects

Z G Evstigneeva

Publications and source records attributed to Z G Evstigneeva.

At least 19 recordsLinked to original sources
PubMed1986

[Inhibition of glutamine synthetase activity by biologically active derivatives of glutamic acid].

The inhibition of activity of glutamine synthetase from Chlorella and porcine brain by 4-hydroxy-D-4-fluoro-D,L- and 4-amino-D,L-glutamic acids diastereoisomers was studied. Each compound was shown to exert the same inhibiting effect on glutamine synthetase from both sources. In case of threo-4-hydroxy-D-glutamic acid the inhibition of the Chlorella enzyme was of a competitive and of a completely mixed type. The enzyme inhibition by 4-fluoro-D, L-glutamic acids seemed to be of a completely non-competitive type. The Ki values for all inhibition reactions were determined. A comparison of biochemical parameters and biological activity revealed that the most effective inhibitors of the enzyme exert a most potent antitumour and antiviral action.

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

[High molecular weight protein consisting of 14 monomers from pea leaves].

A new high molecular weight protein has been detected in pea leaves. Using electron microscopy it has been demonstrated that this protein consists of 14 identical monomers with a point 72 symmetry arranged in two layers, 7 monomers in each. The molecular weight of the protein as determined by gel filtration and sedimentation equilibrium method is equal to 900000 +/- 150000 and 950000 +/- 50000, respectively. The sedimentation coefficient for the protein is 24.3 +/- 1.0S. During SDS polyacrylamide gel electrophoresis the protein dissociates into identical polypeptide chains with molecular weight of 67000 +/- 3000. The circular dichroism spectra of the protein reveal that the percentage of alpha-helix portions, beta-structures, beta-turns and irregular portions is 0.45 +/- 0.06, 0.31 +/- 0.03, 0.09 +/- 0.03 and 0.15 +/- 0.07, respectively. The protein possesses a weak ATPase activity. The protein content in the leaves changes in the course of development.

Circular Dichroism↗
PubMed1982

Multiple molecular forms of glutamine synthetase in pea seeds.

Multiple molecular forms of glutamine synthetase (GS, EC 6.3.1.2) have been studied in pea seeds of different varieties. The number of GS molecular forms in the seeds proved to be related to their colour. Two GS forms in the green seeds have been found and only one of them in the yellow seeds. Green seeds had chlorophyll content amounted to 0.4% of the total pigment content in the leaves. Chloroplasts, somewhat smaller than those in pea leaves of the same variety, have been isolated from green seeds. The presence of the second GS form in the pea green seeds we relate to the chloroplasts. By electrophoretic mobility both forms of GS from the green seeds are not identical to the chloroplast GS and the cytosol GS of leaves. Thus, we believe pea plant to contain, at least, four GS forms.

Chlorophyll↗
PubMed1981

Effect of light on the formation of multiple molecular forms of glutamine synthetase in plants.

The presence of multiple molecular forms (MMF) of glutamine synthetase (GS) has been studied in pumpkin plants and in cotyledons of bean plants. Two MMF of GS have been found in pumpkin leaves and in green cotyledons: chloroplast GS and cytosol GS. Cotyledons of etiolated pumpkin seedlings contain only the cytosol GS. Illumination of etiolated pumpkin seedlings with white light results in the appearance, within one minute, of the second molecular form, the chloroplast GS, which appears to be due to activation rather than de novo synthesis of the enzyme. Cotyledons of resting seeds of horse bean, pea, soybean and lupine contain only one form of GS. The second form, chloroplast GS, appears after germination in the light, but only in those cotyledons of soybean and lupine that can become green.

Chloroplasts↗
PubMed1981

[Electron microscopy of glutamine synthetase from pea leaf chloroplasts].

Using electron microscopy, the spatial structure of glutamine synthetase from pea leaf chloroplasts was studied. The enzyme was shown to consist of eight elongated subunits, which are arranged with a point of 42 symmetry at the vertices of two squares. These squares are twisted about a 4-fold axis at 40 degrees relative to each other.

Chloroplasts↗
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

Electron microscopy of glutamine synthetase from pea leaf chloroplasts.

The structure of pea leaf chloroplast glutamine synthetase was studied by electron microscopy. The enzyme is shown to consist of eight elongated subunits which are arranged with point 42 symmetry at the vertices of two squares. These squares are twisted about the 4-fold axis at 40 degrees relative to each other.

Chloroplasts↗
PubMed1979

[Purification and physico-chemical properties of glutamine synthetase from pea chloroplasts].

A highly purified, practically homogeneous glutamine synthetase was isolated from pea leaf chloroplasts. The enzyme purity was assayed by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The sedimentation coefficient is 16,3S. The sedimentation equilibrium analysis showed that the molecular weight of the enzyme is equal to 480 000. The minimal molecular weights of the enzyme as calculated from the data of polyacrylamide gel electrophoresis in the presence of SDS and the amino acid analysis were found to be 62 000 and 60 000, respectively. The enzyme contains a large amount of dicarboxylic and sulfur-containing amino-acids. The N-terminal amino acid is glycine. The isoelectric point for the enzyme lies within the pH range of 4,2-4-4.

Amino Acids↗
PubMed1978

[Irreversible inactivation of Chlorella glutamine synthetase by urea].

The effect of urea on Chlorella glutamine synthetase (E. C. 6.3.1.2) activity and tertiary structure is investigated. Urea is found to inhibit the activity of glutamine synthetase, the inhibitory effect being independent on the time. The enzyme molecule relax and changes its affinity to ammonium under the effect of urea at concentrations of 1.0-4.0 M. Higher concentrations of urea (5,0 M and more) produce a dissociation of the enzyme molecule into monomers without any intermediate forms. Monomers do not possess any synthetase and transferase activities. Substrates and cofactors do not protect the enzyme from the effect of urea and do not stimulate the emzyme reactivation and reaggregation after its dissotiation. The data obtained are discussed from the viewpoint of the regulation of Chlorella glutamine synthetase activity in vivo.

Animals↗
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↗
PubMed1977

[Purification, properties and quaternary structure of glutamine synthetase from Chlorella].

A highly purified preparation of glutamine synthetase from chlorella grown on a medium containing nitrate as a sole source of nitrogen, was isolated and characterized by disc-electrophoresis and analytical ultracentrifugation. The N-terminal amino acid of glutamine synthetase is glycine. The molecular weight of glutamine synthetase is 32.000; its activity in the presence of Mg2+ was 150 mkmol o-phosphate per min per mg protein. The molecular weight of subunits of the enzyme, equal to 53.000 was determined by disc-electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate. Electron microscopy of negatively contrasted enzyme preparations revealed 6 subunits in the enzyme molecule, arranged in a point symmetry group 32.

Chlorella↗
PubMed1977

[Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components].

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.

Adenine Nucleotides↗
PubMed1976

[Regulation of glutamine metabolism in Chlorella pyrenoidosa. Mechanisms of regulating the activity of glutamine synthetase during ammonia assimilation].

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.

Ammonia↗
PubMed1976

[Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids].

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.

Amino Acids↗