[Comparative study of different variants of Actinomyces roseoflavus producing the polyene antibiotic roseofungin]. / Sravnitel'noe issledovanie razlichnykh variantov Actinomyces roseoflavus, obrazuiushchego polienovyi antibiotik rozeofungin.

The respiration chain in the membranes of whole Actinomyces roseoflavus (var. roseofungini) cells from the parent and secondary cultures is sensitive to KCN, non-sensitive to Triton X-100 treatment removing the antibiotic roseofungin from the cells, and has a very high for the bacteria respiration control. When the cells are in contact with atomic tritium at the temperature of liquid nitrogen, roseofungin is tritiated and binds to A. roseoflavus isolated membranes and whole cells, mostly to those of the parent culture as compared to the secondary culture. A fraction of membranes which lost NADH dehydrogenase in the course++ of purification was isolated from the cells disintegrated in the frozen state.

[Action of the steroid glycoside deltonin on the bacterial membranes of Micrococcus lysodeikticus]. / Deistvie steroidnogo glikozida del'tonina na bakterial'nye membrany Micrococcus lysodeikticus.

A slight detergent-like effect of steroid glycoside deltonine from Dioscorea deltoidea on the bacterial membranes of Micrococcus lysodeikticus was detected which resulted in the breaking of the osmotic barrier of protoplasts and in the loss from the membranes of small fragments containing the dehydrogenases of the respiratory chain but without cytochromes. These small fragments still retained the membrane structure.

[Interaction of NADH-dehydrogenase from M. lysodeikticus membranes with lipids in a reconstituted system]. / Issledovanie vzaimodeistviia NADH-degidrogenazy iz membran M. lysodeikticus s lipidami v rekonstruirovannoi sisteme.

The NADH-dehydrogenase isolated from the M. lysodeikticus membranes was reconstituted into liposomes from the lipids obtained from the same membranes. The presence and degree of the reconstitution were investigated by two-dimensional immunoelectrophoresis and photoreactive hydrophobic label. The quenching of protein fluorescence by the aqueous quencher J- was practically the same for the enzyme in the reconstituted system and in the detergent solution, whereas the quencher interacting with the membrane--cetylpyridinium chloride--was effective in the first case and not effective in the second one. Evidence for the energy transfer from protein chromophores of NADH dehydrogenase in the proteoliposomes (lambda excit = 286 nm) to the hydrophobic fluorescent probe pyrene was obtained. It was found that about 30% of the chromophores in the enzyme molecule are involved in this process. The hydrophobic spin probe, whose paramagnetic fragment is located on the surface and not inside the hydrophobic phase of the membrane, can act as electron acceptor during NADH oxidation in the reconstituted system. The data obtained are suggestive of the exposure of the bulk of the enzyme molecule to the environment and of interaction of the smaller part of the molecule with the lipid phase. The active center is located on the part of the enzyme molecule which is exposed to water. It is assumed that the NADH-dehydrogenase molecule is exposed to water. It is assumed that the NADH-dehydrogenase molecule is involved in heat diffusion which facilitates the active center interaction with the membrane surface.

[Study of membrane proteins from Microccus lysodeikticus using immunochemical methods]. / Izuchenie membrannykh belkov Micrococcus lysodeikticus metodami.

Using immunoelectrophoresis, the antigenicity of various protein fractions of the Micrococcus lysodeikticus membranes was evaluated. It was shown that both the peripheral and integral membrane proteins possess the antigenic determinants. The antiserum exhausted by the M. lysodeikticus mebranes loses its ability to interact with intergral proteins, which are not solubilized by Triton X-100. It was thus assumed that the integral proteins are exposed on the membrane surface constantly or periodically and that there exist no proteins which are completely and permanently incorporated into the lipid bilayer. The respiratory chain of the M. lysodeikticus membrane is inhibited by membrane immunoglobulins by 50%. This is probably due to the presence in the membrane antiserum of antibodies specific to the respiratory chain enzymes. Evidence for this assumption can be derived from the fact that partially purified cytochrome b556 forms a precipitation zone with the membrane antiserum and that the activity of membrane NADH-dehydrogenase is inhibited by a monoserum against NADH-dehydrogenase.

[Bacterial membrane proteins. Properties of Micrococcus lysodeikticus NADH dehydrogenase]. / Belki bakterial'nykh membran. Svoistva NADH-degidrogenazy iz Micrococcus lysodeikticus.

NADH dehydrogenase was isolated from M. lysodeikticus membranes with FAD as a prosthetic group. It was found the enzyme molecular weight is about 140000 in 0,01 M phosphate buffer, pH 7,4 in 1% Triton X-100. The enzyme molecules are dimers consisting of two subunits with molecular weight of 70000. The content of alpha-helical regions is 30%, that of beta-forms is 13%. The protein globule is cross-linked with the disulfide bonds and has hydrophobic regions on its surface.

[Cytochrome b556 complexes solubilized from Micrococcus lysodeikticus membranes by triton X-100]. / Kompleksy tsitokhroma b556, soliubilizirovannye iz membran Micrococcus lysodeikticus tritonom X-100.

The integral protein of cytochrome b556 after its solubilization with Triton X-100 from M. lysodeikticus membranes was studied. The cytochrome was found in complexes differing in charge and size during preparative gel electrophoresis and centrifugation in a sucrose concentration gradient. Cytochrome b556, being in complexes, retains its ability to be reduced by NADH dehydrogenase. The electron micrographs of the membranes after solubilization by Triton X-100 demonstrated the maintenance of the membrane structure. It is concluded that native protein complexes marked with cytochrome b556 are extracted from the membranes under their solubilization.

[Proteins of bacterial membranes. Purification of NADH-dehydrogenase by electrofocusing]. / Belki bakterial'nykh membran ochistka NADH-degidrogenazy iz M. lysodeikticus s pomoshch'iu elektrofokusirovki

A highly purified preparation of NADH dehydrogenase was isolated from bacteria M. lysodeikticus membranes. The purification procedure involved extraction of the enzyme complex from isolated membranes by EDTA, solubilization of the complex by non-ionogenic detergent (1% Triton X-100), chromatography on DEAE-cellulose and electrofocussing in the pH gradient 4-6. The isoelectric point of the preparation is at 4.5; its main component is a protein with m.w. of about 76.000.

[Change in lipid-protein interactions in the membranes of bacteria exposed to gramicidin S]. / Izmenenie lipid-belkovykh vzaimodeistvii v membrane bakterii pri deistvii gramitsidina S

Effect of cyclopeptide antibiotic gramicidin S on some enzymes and physical state of isolated Micrococcus lysodeikticus membranes is studied. Malate and lactate dehydrogenases were monotonously inhibited under the increase of gramicidin S concentration, while the activity of NADH-dehydrogenase firstly decreased and then reversed to the initial level under further increase of gramicidin S concentration. The oxygen uptake under oxidation of NADH and malate with membranes almost completely inhibited by the antibiotic, while the activity of ascorbate-TMPD-oxidase activity slightly inhibited by the same concentration of gramicidin. The addition of Triton X-100 completely eliminated the inhibitory effect of gramicidin on malate dehydrogenase. The introduction into the membrane of spine probes (2,2,6,6-tetramethyl-4-palmitoylamidopiperidine-1-oxile and 2(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxyazolidinyloxile) revealed that gramicidin caused the condensation of membrane lipid component. It is suggested that ionic interaction of gramicidin S with membrane phospholipids brings to "a freezing" of lipids which is a direct cause of impairing the activity of membrane respiration enzymes and the change of their position in the lipid matrix, thus inhibiting energy-producing processes in cell.

[Effect of levomycetin (chloramphenicol) on the biosynthesis of membrane proteins, structure and some functions of Micrococcus lysodeikticus bacterial membranes]. / Deistvie levomitsetina (khloramfenikola) na biosintez membrannkh belkov struktury i nekotorye funktsii membran bakterii Micrococcus lysodeikticus

Levomycetin (chloroamphenicol), an inhibitor of protein synthesis, caused drastic changes in the molecular organization of bacterial membranes being introduced into the cultural medium of Micrococcus lysodeikiticus (50-100 mkg/ml. Isolated membranes of levomycetin-treated cells are enriched with lipids as compared with the control, they are more labile and they lose the X protein component and a considerable part of dehydrogenases, which results in the significant inhibition of all the respiration chain. At the same time the content of cytochrome alpha was increased by 17%, and the activity of malate dehydrogenase (as estimated in cell lysates) was two-fold increased in levomycetin-treated cell as compared with the control. It means that biosynthesis of some membrane proteins can be twice more resistant to the action of the antibiotic than the biosynthesis of other proteins, the total protein biosynthesis being significantly depressed (the incorporation of labelled leucine in membrane proteins is inhibited by 78%, and in cytoplasmic proteins-by 83%). The ratio of the respiration rate of intact levomycetin-treated cells to the protein content or to the cell bulk was similar to that of the control culture, which testifies the stability of membranes in vivo even under considerable "fattening". The loss of dehydrogenases and the X protein from the membranes apparently takes place at the moment of the cell rupture during the lysis in hypotonic medium. The ratio of the respiration rate of intact levomycetin-treated cells to the content of cytochromes a, b, c was 1.15-1.55 times as low as that of control cells which is probably due to the tendency of respiration components to the independent translocation in the membrane and is the result of the decrease in the concentration of the respiration components under the less than fattening greater than of the membrane.