Study of membrane proteins from Microccus lysodeikticus using immunochemical methods]

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.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Effects of Cerulenin upon the Syntheses of Lipid and Protein and upon the Formation of Respiratory Enzymes in Adapting, Lipid-Limited Saccharomyces cerevisiae

When bakers' yeast cells were grown anaerobically in a medium supplemented with Tween 80 and ergosterol, exposure during aeration to the fatty acid synthesis inhibitor, cerulenin, had little effect upon respiratory adaptation, the induction of enzymes of electron transport, or the in vivo incorporation of [(14)C]leucine into mitochondrial membranes. These lipid-supplemented cells were apparently able to undergo normal respiratory adaptation utilizing endogenous lipids alone. The level of cerulenin used (2 mug/ml) inhibited the in vivo incorporation of [(14)C]acetate into mitochondrial membrane lipids by 96%. If, however, the cells were deprived of exogenous lipid during anaerobic growth, subsequent exposure to cerulenin severely reduced their capacity to undergo respiratory adaptation, to form enzymes of electron transport, and to incorporate amino acid into both total cell and mitochondrial membrane proteins. This cerulenin-mediated inhibition of enzyme formation and of protein synthesis was nearly completely reversed by the addition of exogenous lipid during the aeration of the cells. In lipid-limited cells, chloramphenicol also had dramatic inhibitory effects, both alone (75%) and together with cerulenin (85%), upon total cell and mitochondrial membrane [(14)C]leucine incorporation. This marked chloramphenicol-mediated inhibition was also largely reversed by exogenous lipid. It is concluded that, in lipid-limited cells, either cerulenin or chloramphenicol may prevent the emergence of a pattern of lipids required for normal levels of protein synthetic activity. The effect of cerulenin upon the formation of mitochondrial inner membrane enzymes thus appears to reflect a nonspecific effect of this antilipogenic antibiotic upon total cell protein synthesis.     

Lipid fluidity and membrane protein dynamics

Membrane fluidity plays an important role in cellular functions. Membrane proteins are mobile in the lipid fluid environment; lateral diffusion of membrane proteins is slower than expected by theory, due to both the effect of protein crowding in the membrane and to constraints from the aqueous matrix. A major aspect of diffusion is in macromolecular associations: reduction of dimensionality for membrane diffusion facilitates collisional encounters, as those concerned with receptor-mediated signal transduction and with electron transfer chains. In mitochondrial electron transfer, diffusional control is prevented by the excess of collisional encounters between fast-diffusing ubiquinone and the respiratory complexes. Another aspect of dynamics of membrane proteins is their conformational flexibility. Lipids may induce the optimal conformation for catalytic activity. Breaks in Arrhenius plots of membrane-bound enzymes may be related to lipid fluidity: the break could occur when a limiting viscosity is reached for catalytic activity. Viscosity can affect protein conformational changes by inhibiting thermal fluctuations to the inner core of the protein molecule.     

Biochemical studies of the chromaffin granule. III. Redistribution of lipid phosphate,dopamine-β-hydroxylase and chromogranin a after freezing and thawing of the isolated granule membranes

Freezing and thawing a dialysed suspension of lysed chromaffin granules and sedimented membrane preparations resulted in redistribution of lipid phosphate and protein. By this treatment the high ratios of lipid phosphate/protein in the membrane fragments, isolated on sucrose density gradient from the dialysed suspensions and the sedimented membrane preparation, were reduced from 1.56 to 1.03 μmoles/mg and from 1.97 to 0.83 μmoles/mg, respectively.Multilamellar, liposomal structures could be isolated from the frozen and thawed membrane preparations and were found to sediment in the 0.4 M sucrose layer by density gradient centrigugation. This fraction was without morphological resemblance to the intact chromaffin granules or their membranes and was found to account for 53% of the lipid phosphate, 35% of chromogranin A, 21% of dopamine-β-hydroxylase activity and 12% of the protein of the total preparation. The specific activities of chromogranin A and dopamine-β-hydroxylase in the artificially formed liposomal structures closely resembled that of the solubilized protein and was significantly higher than in the lipid phosphate-depleted membrane fragments recovered in the 1.1 M sucrose layers.It is concluded that freezing and thawing as a means of purifying the isolated granule membranes lead not only to the solubilization of chromogranin A, but also to removal of dopamine-β-hydroxylase activity and lipid phosphate from the labile membrane fragments.     

Kinetics of integrated electron transfer in the mitochondrial respiratory chain: random collisions vs. solid state electron channeling

Recent evidence, mainly based on native electrophoresis, has suggested that the mitochondrial respiratory chain is organized in the form of supercomplexes, due to the aggregation of the main respiratory chain enzymatic complexes. This evidence strongly contrasts the previously accepted model, the Random Diffusion Model, largely based on kinetic studies, stating that the complexes are randomly distributed in the lipid bilayer of the inner membrane and functionally connected by lateral diffusion of small redox molecules, i.e., coenzyme Q and cytochrome c. This review critically examines the experimental evidence, both structural and functional, pertaining to the two models and attempts to provide an updated view of the organization of the respiratory chain and of its kinetic consequences. The conclusion that structural respiratory assemblies exist is overwhelming, whereas the expected functional consequence of substrate channeling between the assembled enzymes is controversial. Examination of the available evidence suggests that, although the supercomplexes are structurally stable, their kinetic competence in substrate channeling is more labile and may depend on the system under investigation and the assay conditions. mitochondria; supercomplexes; ubiquinone; complex I (NADH-ubiquinone oxidoreductase)     

Changes in properties of the inner mitochondrial membrane during mitochondrial biogenesis in aging sweet potato tissue slices in relation to the development of cyanide-insensitive respiration.

Protein and phospholipid of the inner mitochondrial membrane in sweet potato root tissue increased after a lag phase during aging of the sliced tissue. The protein, but not the phospholipid, from aged slices was more insoluble in a solution containing sodium deoxycholate and sodium cholate than that from fresh tissue. There were differences in polypeptide composition between deoxycholate-cholate-soluble and -insoluble fractions as determined by polyacrylamide-gel electorophoresis of membrane fragments in the presence of sodium dodecyl sulfate. However, no difference was observed between mitochondrial membranes from fresh and aged slices. When disrupted mitochondrial membrane was subjected to equilibrium density centrifugation, two bands were obtained from aged slices but only one band from fresh tissue. The lighter band from aged slices was indentical to the single band from fresh tissue. The denser band was very poor in phospholipid, and the protein was very insoluble in deoxycholate-cholate solution. The denser membrane fragments possessed a cyanide-insensitive respiratory chain whereas the lighter did not. It is proposed that the development of the cyanide-insensitive respiration in aging slices is related to the formation of phospholipid-deficient mitochondrial membrane.     

Lateral diffusion of the protein components of the respiratory assembly of Microsoccus lysodeikticus]

Preparations with a selectively decreased (by 85-90%) content of NADH dehydrogenase were isolated by means of heating treatment of M. lysodeikticus isolated membranes. The degree of the reduction of the NADH dehydrogenase nearest neighbour in the respiration chain of cytochrome b556 in heated membranes is similar to that in intact membranes. It is concluded that cytochrome b556 and (or) NADH dehydrogenase are capable to lateral migration in the membrane of M. lysodeikticus, resulting in the inter-chain electrone transport. A coefficient of their lateral diffusion is calculated (D equals 8-10(-10)-2-10(-9) CM2SEC-1 At 30 degrees C) on the basis of kinetics of cytochrome reduction by NADH dehydrogenase. The electron transport, due to a diffusion of respiration carriers from one assambly to another, proceeds 100 times as slow as the electrone transport in the respiratory chain. The data obtained allow to consider the aggregation of respiration enzymes as a dynamic formation.     

Stimulation of chloride transport by fatty acids in corneal epithelium and relation to changes in membrane fluidity.

The effect of altering cell membrane lipids on ion transport across isolated corneas was studied. Corneas mounted in Ussing-type chambers showed a rapid increase in short-circuit current following treatment with a variety of unsaturated fatty acids of varying chain length and unsaturation. Measurements of membrane fluidity which utilize immunofluorescence labelling of membrane proteins showed corneal epithelial cell membranes to be significantly more fluid following linoleic acid treatment. Uptake studies indicate rapid incorporation of [14C]linoleic acid into corneal cell membranes. Highly unsaturated fatty acids were found to have the greatest ability to stimulate chloride transport. Saturated fatty acids were tested and were found to have no effect on chloride transport at any concentration. It is proposed that unsaturated fatty acids activate chloride transport by increasing membrane lipid fluidity. The relationship of these parameters is discussed in terms of a mobile receptor model. We speculate that an increase in membrane lipid fluidity promotes lateral diffusion of membrane receptor proteins and enzymes, increasing protein-protein interactions within the membrane, ultimately resulting in the enhancement of cyclic AMP synthesis.     

Membranes of bacteria and mechanism of action of the antibiotic gramicidin S]

The cyclopeptide antibiotic gramicidin S taken at a concentration of 100--200 mkg/mg membrane protein rapidly increases the permeability of M. lysodeikticus protoplast membranes for substrates of respiratory chain and exogenous cytochromes c. Prolonged incubation of gramicidin S with protoplasts results in their lysis which is more fast at low temperatures. In contrast to natural gramicidin, a derivative of gramicidin S with acetylated amino groups does not inhibit either the micrococcus membrane dehydrogenase or the whole of respiratory chain and does not affect the osmotic barrier of protoplasts. Aliphatic diamines (at concentrations up to 0.1 M) and Ca2+ ions (10(-2) M) do not affect the functioning of the respiratory chain in isolated micrococcus membranes. Another derivative of the antibiotic with an increased distance of loaded amino groups from the cyclopeptide framework (diglycyl gramicidin S) affects the membrane in a way similar to that of natural gramicidin. Washing of gramicidin-treated membranes with NaCl enhances the inhibitory effect of the antibiotic on membrane enzymes. The data obtained suggest that in addition to ionic interactions some hydrophobic interactions also occur during gramicidin S binding to the bacterial membrane, probably at the expense of a hydrophobic peptide ring. It is assumed that gramicidin S, similar to Ca2+ and some other membranotropic agents provides for phase separation of negatively charged phospholipids from other groups of phospholipids, manifesting itself in an appearance of "frozen" sites on the membrane which destroys its barrier properties. This is due to the formation of ionic bonds of negatively charged phospholipids. Simultaneously, unlike Ca2+, gramicidin S, when interacting with membrane proteins, prevents their redistribution in more liquid parts of the membrane, which results in a situation when the respiratory enzymes become surrounded by alkyl chains with restricted motion.     

THE ISOLATION OF RETINAL OUTER SEGMENT FRAGMENTS

Bovine retinal outer segment fragments were isolated by density gradient centrifugation in a high centrifugal field. Assays of the final preparation for enzymes of the mitochondrial respiratory chain indicated mitochondrial contamination not in excess of 1 per cent. Glucose-6-phosphatase and TPNH-cytochrome c reductase activities, presumably diagnostic for microsomes, were also absent. Electron micrographs did not disclose the presence of significant numbers of particles other than fragments of the outer segment discs. The red fragments were characterized by an ascorbate-oxidizing system and a high lipid content.     

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.     

Characterization of cellular and extracellular plasma membrane vesicles from a non-metastasizing lymphoma (Eb) and its metastasizing variant (ESb)

Plasma membrane fragments from two variants of a murine lymphoma, Eb and ESb, with different metastatic capacity were investigated. Plasma membranes were isolated from tumor cells recovered from the peritoneal cavity. They differed in their lipid composition, indicating a more fluid state of the plasma membranes derived from the highly metastatic tumor line ESb. Extracellular membrane vesicles could be isolated from the ascites of the tumor-bearing mice. The shedding capacity of ESb cells was much higher than that of Eb cells. The extracellular membranes by chemical analysis and the measurement of marker enzymes proved to be derived from the plasma membranes. However, they differed from the plasma membranes from which they were derived in several aspects: (i) the lipid to protein ratio was diminished; (ii) the activities of some plasma membrane-associated enzymes were lower while other were identical in plasma membranes and extracellular membranes; (iii) the content of saturated fatty acids in phopholipids was enhanced in extracellular membranes. These effects were more pronounced in the highly metastasizing tumor line ESb. It is thus concluded that shedding of extracellular membranes is not a random process. The biochemical differences found in the plasma membranes and the extracellular membranes of the two tumor lines are discussed with respect to the different metastatic capacity of the tumors.     

The effects of lipid fluidity on the rotational diffusion of complex I and complex III in reconstituted NADH-cytochrome c oxidoreductase

NADH-ubiquinone oxidoreductase (Complex I) can be recombined with ubiquinol-cytochrome c oxidoreductase (Complex III) to reconstitute NADH-cytochrome c oxidoreductase. Two modes of interaction have been found. In one, the Complexes interact stoichiometrically in one to one molar ratios to give a binary Complex I-III unit. In the other, the kinetics of NADH-cytochrome c oxidoreductase are characteristic of 'Q-pool' behaviour seen in intact mitochondria and submitochondrial particles in which the Complexes need not interact directly but can do so via a pool of mobile ubiquinone. Stoichiometric behaviour is found when only boundary layer or annular lipid is present or the lipid is in the gel phase. The lipid is immobile on the ESR time scale and protein rotational diffusion, measured by saturation transfer ESR, is very slow. Q-pool behaviour is found when mobile extra-annular lipid phase is also present. Protein rotational diffusion is rapid and characteristic of a fully disaggregated state. We have also used freeze-fracture electron microscopy of reconstituted NADH-cytochrome c oxidoreductase to monitor protein aggregation and lateral phase separation of lipids and proteins under various conditions. We discuss our findings in relation to models for lateral interactions between respiratory chain enzymes.     

Photoaffinity labelling of submitochondrial membranes with the 3-azido analogue of 9-amino-3-chloro-7-methoxyacridine

9-Amino-3-azido-7-methoxyacridine has been synthesized and shown to be a suitable photoaffinity probe for the site(s) of interaction of 9-amino-3-chloro-7-methoxyacridine with submitochondrial membranes. Both the excitation and emission spectra of the azido analogue covalently bound to membranes in the energized state display distinctive differences from the spectra of labelled, non-energized membranes (i.e., in the absence of oxidizable substrate, or its presence when uncoupler (FCCP) is also present during photolysis). Enzymatic analyses indicate that the probe interacts with the ATPase and the respiratory chain enzymes; energization appears to afford some protection against inactivation. Electrophoresis of the labelled membranes and isolation of their lipid and protein components indicate that the spectral differences are attributable to differing interactions with the lipid components of energized, relative to non-energized, membranes. Similar results have been obtained with the 3-azido analogue of quinacrine (Mueller, D.M., Hudson, R.A. and Lee, C.P. (1982) Biochemistry 21, 1445-1453), which differs significantly, however, in the extent of its interactions with the enzymes of the respiratory chain and the ATPase. These results indicate that the energy-linked fluorescence responses of 9-aminoacridines with submitochondrial membranes arise from direct interactions with membrane components and may involve redistribution of the probe molecules and/or alteration of their microenvironments upon energization.     

Biochemical and morphological changes in dormant (“Nonculturable”) <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> cells

Biochemical and morphological changes have been studied during transition of Mycobacterium smegmatis cells into their dormant (“nonculturable”) state. A significant fraction of the population of irreversibly “nonculturable” (NC) cells has a thicker cell wall, condensed cytoplasm, and a decreased number of ribosomes. The lipid contents in the NC cells are lower than in the metabolically active cells, with a relatively decreased amount of phospholipid and neutral lipid. Free mycolic acids, which are abundant in metabolically active cells, were not found in the NC cells. The NC forms are also characterized by decreased respiratory activity on endogenous substrates; however, the respiratory chain enzymes retain their activities in the isolated membranes. Activities of the Krebs cycle and glyoxylate cycle enzymes are markedly decreased. Despite a significant decrease in metabolic activity, NC cells possess membrane potential that seems to provide for reversibility of the NC state of mycobacteria, i.e. their capability of reactivating.     

Sticholysin I–membrane interaction: An interplay between the presence of sphingomyelin and membrane fluidity

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.     

A role for Tim21 in membrane-potential-dependent preprotein sorting in mitochondria

The mitochondrial inner membrane harbors complexes of the respiratory chain and translocase complexes for preproteins. The membrane potential generated by the respiratory chain is essential for ATP production by the mitochondrial ATP synthase and as a driving force for protein import. It is generally believed that the preprotein translocases just use the membrane potential without getting into physical contact with respiratory-chain complexes. Here, we show that the presequence translocase interacts with the respiratory chain. Tim21, a specific subunit of the sorting-active presequence translocase , recruits proton-pumping respiratory-chain complexes and stimulates preprotein insertion. Thus, the presequence translocase cooperates with the respiratory chain and promotes membrane-potential-dependent protein sorting into the inner mitochondrial membrane. These findings suggest a new coupling mechanism in an energy-transducing membrane.     

运动性疲劳状态下大鼠心肌线粒体内膜变化的研究

采用递增负荷力竭性运动模型,观察了Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠急性运动至力竭后心肌线粒体内膜流动性、ＮＡＤＨ－ＣｏＱ还原酶及ＡＴＰ酶活性的变化。结果表明,大鼠心肌线粒体内膜荧光偏振值较安静时显著增高（Ｐ＜０．０１）,示膜流动性降低。线粒体内膜ＮＡＤＨ－ＣｏＱ还原酶和肌线粒体内膜功能改变,其膜流动性和呼吸链酶活性变化,可能是运动性疲劳的重要膜分子制之一。     

Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature'). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.