Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus.

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the period of decelerating growth.     

Isolation and properties of mesosomal membrane fractions from Micrococcus lysodeikticus

1. A method is described for the isolation of pure mesosomal membrane fractions from Micrococcus lysodeikticus. 2. Plasmolysis of cells, before wall digestion, was necessary for effective mesosome release. 3. The effect of mild shearing forces, temperature and time upon the release of mesosomal membrane from protoplasts was investigated. 4. The optimum yield of mesosomal membranes from stable protoplasts was achieved at 10mm-Mg(2+). 5. Mesosomal membrane vesicle fractions prepared at differing Mg(2+) concentrations above 10mm were similar in chemical composition. 6. Comparison of the properties of peripheral and mesosomal membrane fractions revealed major differences in the distribution of protein components, membrane phosphorus, mannose and dehydrogenase activities between the two fractions. 7. Only cytochrome b(556) was detected in mesosomal membranes, whereas peripheral membranes contained a full complement of cytochromes. 8. Preliminary investigations suggested the localization of an autolytic enzyme(s) in the mesosomal vesicles. 9. The anatomy of mesosomal and peripheral membrane have been compared by the negative-staining and freeze-fracture technique. 10. The results are discussed in relation to a plausible role for the mesosome.     

Bacterial membrane proteins. Properties of Micrococcus lysodeikticus NADH dehydrogenase]

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.     

Restoration of deoxycholate-disrupted membrane oxidases of Micrococcus lysodeikticus

Membrane-associated l-malate and reduced nicotinamide adenine dinucleotide (NADH) oxidase complexes of Micrococcus lysodeikticus were inactivated with deoxycholate. Reactivation of NADH oxidase by addition of Mg(2+) occurred in these detergent-membrane mixtures, but reactivation of l-malate oxidase did not occur in the presence of deoxycholate. Removal of detergent by gel filtration allowed Mg(2+)-dependent restoration of both l-malate and NADH oxidases. Maximal NADH and l-malate oxidase restoration required 10 min and 40 min, respectively, at 30 mm MgSO(4). Maximal restoration of both oxidases required at least 12 mm MgSO(4) in an incubation period of 1 hr. Reduced-minus-oxidized difference spectra of Mg(2+)-restored membrane oxidases showed participation of cytochromes b, c, and a when either l-malate or NADH served as reductant; addition of dithionite did not increase the alpha- and beta-region absorbancy maxima of these hemoproteins when restored membranes were first reduced with the physiological substrates l-malate or NADH. Not all divalent cations tested were equally effective for reactivation of both oxidases. l-Malate oxidase was restored by both Mn(2+) and Ca(2+). NADH oxidase was not activated by Mn(2+) and only slightly stimulated by Ca(2+). Separation of deoxycholate-disrupted membranes (detergent removed) into soluble and particulate fractions showed that both fractions were required for Mg(2+)-dependent oxidase activities. Electron micrographs indicated conditions of detergent treatment did not destroy the vesicular nature of protoplast ghost membranes.     

Osmotic water permeability and solute reflection coefficients of rat kidney brush-border membrane vesicles

Solute reflection coefficients, sigma i, of rat kidney brush-border membrane vesicles were determined by the comparison of water flows induced by equiosmolal gradients of sucrose and NaCl, KCl or mannitol. The values of 0.53 for sigma NaCl and 0.56 for sigma KCl when compared with 0.92 for sigma mannitol suggested some interactions between salt and water pathways. Altering the membrane proteins with 0.4 mM HgCl2 decreased the osmotic water permeability of the vesicles by 70 to 80% and brought sigma NaCl and sigma KCl to a value not different from 1. This argued in favor of water protein pathways in the luminal membrane of kidney proximal cells which are partly accessible to NaCl and KCl.     

Partial characterization of membrane-associated proteinases from Micrococcus lysodeikticus

Summary We have identified cytoplasmic and membrane-associated proteinases from Micrococcus lysodeikticus (M. luteus) by the use of ¹²⁵I-labeled casein and insulin as substrates. The membrane-associated activities were released by shock washing. Proteolytic activities showed pH optima at slightly alkaline values and we have concentrated on the activities at pH 8.0. The total units of both proteolytic activities were higher in the cytoplasmic than in any other fractions but the situation was different when the results were expressed in terms of specific activity. The activities against casein and insulin were differentiated by the action of inhibitors, divalent metal ions, Arrhenius plots and dependence on ionic strength. On these grounds, it is proposed that the membrane-associated enzyme acting on insulin is a single thiol proteinase while the proteolysis of casein reflects the action of, at least, two enzymes (thiol proteinase and serine proteinase). The distinction between the casein and insulin degrading activities was confirmed by crossed-inhibition experiments and by their behaviour on gel chromatography and concentration-dependent experiments.The aggregating properties have hampered the purification of the enzymes. The present results raise doubts about the significance of preventing membrane damage and degradation of membrane proteins by the addition of indiscriminated proteinase inhibitors during membrane isolation and manipulation.