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G V Tikhonova

Publications and source records attributed to G V Tikhonova.

At least 19 recordsLinked to original sources
PubMed1983

[Submergence of Micrococcus lysodeikticus F1-ATPase into the hydrophobic phase of the membrane, using 2,4,6-trinitrobenzosulfonate and 12-0-(azidoformyl) stearic acid methyl ester].

The accessibility of F1-ATPase from Micrococcus lysodeikticus in solution and in the membrane for the specific water-soluble NH2-group reagent, 2,4,6-trinitrobenzosulfonate (TNBS), was studied. Incubation of the soluble factor F1 with 50 mM TNBS pH 8.3 results in incorporation of 58.6 +/- 4.4 trinitrophenyl residues per mole of enzyme. At the same time F1-ATPase isolated from TNBS-pretreated membranes contains 27.2 +/- 2.0 TNP-residues per mole of enzyme. It is assumed that the different accessibility of F1-ATPase for TNBS in solution and in the membrane is due to incorporation of F1-ATPase into the membrane. Study of membrane F1-ATPase interaction with the radioactive lipid-soluble photoreactive label, 12-0-(azidoformyl) stearic acid methyl ester demonstrated that F1-ATPase does not immediately interact with the lipid phase of the membrane. It is suggested that membrane F1-ATPase may be enveloped by hydrophobic proteins.

Affinity Labels↗
PubMed1983

[Characteristics of subunit composition of H+-ATPase from the anaerobic bacterium Lactobacillus casei].

In the presence of Mg2+ or Ca2+ the membranes of the anaerobic glycolytic bacterium Lactobacillus casei hydrolyze 0.1-0.2 mumole ATP/min/mg of protein with a pH optimum 6.4. This activity is inhibited by N,N'-dicyclohexylcarbodiimide and is insensitive to oligomycin, ouabain, vanadate and hydroxylamine. A soluble ATPase was isolated and purified from L. casei membranes. The specific activity of this ATPase is 3.0-4.0 mumole ATP/min/mg of protein. The enzyme homogeneity was established by analytical polyacrylamide gel disc electrophoresis and by analytical centrifugation (S20, omega = 12 +/- 0,5). The molecular weight of the enzyme is 270 000. Polyacrylamide gel electrophoresis of ATPase denaturated by 1% SDS and 8 M urea in the presence of SDS revealed one type of subunits with Mr = 43 000. These subunits could not be separated by isoelectrofocusing in polyacrylamide gel in the presence of 8 M urea and migrated as a single peptide with pI at 4.2. The experimental results suggest that the soluble ATPase from L. casei consists of six identical subunits with Mr of 43 000.

Anaerobiosis↗
PubMed1983

[Generation of membrane potential by aerobic bacteria Micrococcus lysodeikticus. Correlation between coupled and uncoupled respiration].

The effects of cyanide and nonylhydroxyquinoline-N-oxide on membrane potential generation and oxidase activities in intact cells and membrane particles of M. lysodeikticus were studied. Low concentrations of the inhibitors interacting with the components of one branch of a branched respiratory chain of M. lysodeikticus strongly inhibit the membrane potential generation and only slightly reduce the total respiration rate. It was assumed that the generation of delta psi takes place in the b-c locus of the respiratory chain branch terminated with cytochrome oxidase; over 90% of total O2 uptake provides for the uncoupled respiration via cytochrome omicron. Inside the b-c locus two sites responsible for delta psi generation were singled out; these sites differ in their sensitivities towards nonylhydroxyquinoline-N-oxide. Based on the data obtained and the effect of "extra-reduction" of cytochrome b566 a hypothetical model of the respiratory chain of M. lysodeikticus was developed, which includes the branching of the electron flux to two terminal pathways and redox equivalent transport cyclization via the menaquinone cycle. The physiological role of uncoupled respiration through cytochrome omicron is discussed.

Aerobiosis↗
PubMed1978

Manipulation of phospholipid composition of membranes with the aid of lipid exchange proteins. Incorporation of phosphatidylcholine into protoplasts of Micrococcus lysodeikticus.

Incubation of Micrococcus lysodeikticus protoplasts with phosphatidylcholine liposomes and rat liver exchange proteins (pH 5.1 supernatant fraction) resulted in replacement of about one half of the bacterial total phospholipids by phosphatidylcholine. Protoplasts modified by phosphatidylcholine showed a decreased rate of oxidation of exogenous substrates (NADH, malate) and decreased ferricyanide reductase activity as compared to the initial protoplasts. At the same time incorporation of phosphatidylcholine had no influence on the level of endogeneous respiration. Protoplasts modified by phosphatidylcholine were osmotically more stable than the initial protoplasts. After osmotic lysis of the phosphatidylcholine protoplasts their NADH (malate) oxidase and ferricyanide reductase activities were restored. Incorporation of phosphatidylcholine into membrane ghosts, obtained by osmotic rupture of the initial protoplasts had only small if any effect on the malate and NADH oxidase and dehydrogenase activities. It is concluded that phosphatidylcholine in incorporated predominantly into the outer part of cytoplasmic membrane and that proteinmediated transfer of phosphatidylcholine results in restoration of the permeability barrier due to repair of local defects in the initial protoplast membrane.

Cell Membrane↗
PubMed1978

[Has the respiratory chain of Micrococcus lysodeiktocus any redox components on the outer surface of cytomembrane?].

M. lysodeikticus protoplasts have catalyzed the reduction of 5.10(-4) M ferricianide by endogenous substrates if the respiratory chain is inhibited by cyanide or anaerobiosis. A disturbance of the protoplast permeability by osmotic shock or Triton X-100 treatment resulted in the decrease of the endogenous ferricianide reduction rate and in simultaneous stimulation of malate ferricianide reductase activity in dehydrogenase site of the inner membrane surface. Reactivation of endogenous ferricianide reduction by protoplasts and the loss of malate stimulating activity were observed in hyperosmotic medium. Unlike malate oxidation by osmotically shocked protoplasts, endogenous protoplast repiration was resistant to ferricianide 5.10(-4) M). The latter, being added to protoplasts, induced the oxidation of anaerobically reduced cytochromes b556+560, and it practically did not affect cytochromes c552 and a601. The data obtained suggest that at least one of the respiratory chain components is arranged on the outer side of the protoplast membrane. This component is probably located in the cytochrome region. The results obtained confirm the hypothesis on transmembrane organization of the respiratory chain in M. lysodeikticus.

Cell Membrane↗
PubMed1977

[Variation of cell membrane lipid composition by means of lipid transfer proteins. Properties of the protoplast membrane of Micrococcus lysodeikticus after incorporation of phosphatidylcholine].

After incorporation of phosphatidylcholine (PC) into the protoplast membrane of M. lysodeikticus by protein mediated transfer from PC liposomes, the activity of some membrane bound respiratory chain enzymes was studied. It was found that incorporation of PC decreases the rates of oxidation of exogenous substrates (NADH, malate) but the level of endogenous respiration was not changed. Ferricyanidreductase activity of ghosts of M. lysodeikticus was not dependent upon the PC content of protoplasts. PC containing protoplasts showed a higher osmotic stability than unmodified protoplasts. It is concluded that the incorporation of PC into the protoplasts results in resealing, i. e. in the repair of local defects in the protoplast membrane.

Cell Membrane↗
PubMed1976

Membrane-reversible H+-ATPase from Micrococcus lysodeikticus.

Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M. lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Adenosine triphosphatase from the membrane of Micrococcus lysodeikticus].

A preparation of ATPase with a high specific activity was isolated from the membrane of M. lysodeikticus. The enzyme was studied using UV-spectroscopy and circular dichroism. The homogeneity of the protein preparation was shown by gel electrophoresis. The catalytic properties of the enzyme were studied using steady state kinetic methods. The values of Km app. and kcat were determined to be 6-10(-4) and 6 mumoles/mg/min respectively. It is shown that ADP is an effective inhibitor of the ATPase reaction, and the inhibition activity increases in the presence of an excess of Ca2+. The nature of the rate dependence of the ATPase reaction on the concentration of the substrate and on Ca-ADP corresponds to a competitive type of inhibition with binding several molecules of Ca-ADP in the active site of the enzyme.

Adenosine Diphosphate↗