Localization of D-lactate dehydrogenase in membrane vesicles prepared by using a french press or ethylenediaminetetraacetate-lysozyme from Escherichia coli.

The localization of D-lactate dehydrogenase in membrane vesicles prepared from Escherichia coli was studied using antibody against the purified enzyme. The activity of D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by using a French press were completely inhibited by this antibody, suggesting that the enzyme is localized on the outside of these vesicles. This and previous results (Futai, 1974) strongly indicate the inversion of these vesicles. The D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by treatment with ethylenediaminetetraacetate-lysozyme were inhibited about 15% by the antibody, whereas proline transport of the vesicles was insensitive to antibody. These results suggest that most of the membrane vesicles have D-lactate dehydrogenase on the inside of the membrane and that such vesicles transport amino acids. This essentially confirms the results of Short, Kaback, and Kohn (1975). However, unlike them we observed that a small but significant portion of activity was sensitive to the antibody as shown above. This portion may represent the completely inverted vesicles in the preparation. Ferricyanide reductase activity cannot be detected in spheroplasts, but about 30 to 50% of the total was detected in membrane vesicles prepared by treatment with ethylenediaminetetraacetate. This confirms our previous findings with membrane prepared by a slightly different procedure. It is concluded that in these vesicles about half the reactive sites for ferricyanide are moved from inside to outside the membrane, whereas 85% of the D-lactate dehydrogenase remains inside the membrane.     

Asymmetric distribution of nitrate reductase subunits in the cytoplasmic membrane of Escherichia coli: evidence derived from surface labeling studies with transglutaminase.

The enzyme transglutaminase has been used to label surface proteins of Escherichia coli cytoplasmic membranes by covalently attaching to them a small fluorescent primary amine, dansyl cadaverine. Spheroplasts lacking outer membrane, osmotically lysed vesicles from the spheroplasts, and vesicles made by breaking cells in a French pressure cell were each labeled with transglutaminase and dansyl cadaverine. When the total cytoplasmic membrane proteins of each were examined on sodium dodecyl sulfate gels, three rather different labeling patterns were obtained. Labeling of the respiratory enzyme, nitrate reductase, in the membranes of each of these preparations was also examined. Membrane-bound nitrate reductase contains three subunits: A, B, and C. Dansyl cadaverine labeling of nitrate reductase in the presence of Triton X-100 indicated that subunits A and C could be labeled. When nitrate reductase was isolated from dansyl cadaverine-labeled spheroplasts, none of the subunits was labeled. When nitrate reductase was isolated from French press vesicles, subunit A was labeled and labeling was enhanced by the presence of nitrate during labeling. When nitrate reductase from osmotic vesicles was examined, subunit A was labeled in the presence of nitrate but no labeled subunits appeared when the vesicles were labeled in the absence of nitrate. It was concluded that (i) nitrate reductase is buried in the membrane with subunit A exposed only on the inner surface of the membrane, (ii) subunit C is sufficiently buried within the membrane so that it is inaccessible to transglutaminase, (iii) subunit B is not labeled under any condition, so its location is not known, and (iv) large osmotic vesicles are probably mosaics in which some protein components have been reoriented.     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts,spheroplast membrane vesicles and chromatophores

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K⁺ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures (“chromatophores”) were obtained by treating spheroplast membrane vesicles by French press or sonication.The membrane structures with either sidedness showed the same light-induced change of the “red shift” type. However, the absorbance change by K⁺ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, “blue shift” in the former and “red shift” in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts, spheroplast membrane vesicles and chromatophores.

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K+ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures ("chromatophores") were obtained by treating spheroplast membrane vesicles by French press or sonication. The membrane structures with either sidedness showed the same light-induced change of the "red shift" type. However, the absorbance change by K+ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, "blue shift" in the former and "red shift" in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Orientation of chromatophores and spheroplast-derived membrane vesicles of Rhodopseudomonas sphaeroides: analysis by localization of enzyme activities.

Intact spheroplasts, vesicles obtained from French-press lysates (chromatophores), and spheroplast-derived vesicles were isolated from photosynthetically grown cells of Rhodopseudomonas sphaeroides. Lysed spheroplasts showed specific activities of succinate, NADH, and l-lactate dehydrogenase which were eight-, six-, and seven-fold higher, respectively, than those of intact spheroplasts when ferricyanide was used as electron acceptor. Mg²⁺-ATPase activity of lysed spheroplasts, measured using an assay system coupled to the oxidation of NADH, was seven-fold higher than the activity of intact sheroplasts. Toluene-treated spheroplast-derived vesicles displayed higher succinate dehydrogenase (ferricyanide reduction) and Mg²⁺-ATPase activities than untreated vesicles whereas no differences were measured between untreated and toluene-treated chromatophores. However, NADH dehydrogenase (ferricyanide reduction) activities of both toluene-treated vesicles and chromatophores were higher than the activities of untreated vesicles and chromatophores. When chromatophores and spheroplast-derived vesicles were preincubated with trypsin, the l-lactate and succinate dehydrogenase activities of chromatophores were preferentially inactivated when phenazine methosulfate was used as electron acceptor. The data indicate that chromatophores are oriented in an opposite direction to the spheroplast-derived vesicles. At least 80% of the latter are oriented in a direction equivalent to the cytoplasmic membrane of intact cells and spheroplasts. Spheroplast-derived vesicles from cells grown with higher light intensities seem to be more uniformly oriented than those obtained from cells grown with lower light intensities.     

Effect of lysophosphatidylcholine on the NADH-ferricyanide reductase activity associated with the plasma membranes of corn roots

Plasma membranes from corn roots (Zea mays L.) were isolated by aqueous two-phase partitioning. A fraction enriched in a vanadate-sensitive ATPase showed characteristics of a plasma membrane ATPase. The sidedness of these vesicles was 89% right-side-out, as evaluated by the ATPase latency. A NADH-ferricyanide reductase was associated with these plasma membrane vesicles. The rate of ferricyanide reduction was 1.3 μmol · min⁻¹·mg⁻¹ protein and was strongly enhanced by the addition of lysophosphatidylcholine (LPC). The effect of this detergent on membrane solubilization and reductase activity was particularly studied. This type of detergent treatment revealed two pH optima (7.0 and 5.0) for the reductase activity, which exhibited biphasic kinetics in the absence or presence of the detergent. These data suggest that two or more reductases could be involved. In addition, membrane vesicle solubilization and determination of ATPase and reductase latency were simultanously studied. From these experiments, it is postulated that the reductase, which exhibits an optimum pH at 7.0 and is slightly stimulated by LPC, could be located on the external side of the plasmalemma. In contrast, the reductase at pH 5.0 strongly stimulated by the detergent treatment, is probably located on the internal side of the membrane, such as the catalytic site of ATPase. Finally, a possible direct action of LPC on the enzymes, is discussed.     

Orientation of NAHD-linked ferric chelate (turbo) reductase in plasma membranes from roots ofPlantago lanceolata

Summary Plasma membrane vesicles isolated from roots ofPlantago lanceolata L. revealed approximately 70% right-side-out orientation based on structure-linked latency with H⁺-ATPase as a marker. Incubation with 0.05% Brij 58 caused the formation of sealed insideout vesicles, evidenced by assaying ATP-dependent proton pumping activity with the optical pH probe acridine orange. NADH-linked FeEDTA reductase activity was stimulated by including either Triton X-100 or Brij 58 in the assay medium. The activity of inverted (Brijtreated) vesicles was not further increased by the addition of Triton, suggesting that maximum activity was obtained in inside-out vesicles. Iron deficiency resulted in a ca. 2-fold increase in the specific activity of both ATPase and Fe(III) chelate reductase but did not cause significant alterations with respect to the effect of detergents. It is concluded that in vitro both donor and acceptor sites of NADH-FeEDTA reductase are located on the cytosolic face of the membrane and trans-oriented flow of electrons is not detectable in plasma membrane vesicles. Unlike Fe chelate reduction in vivo, the plasma membrane-bound reductase activity was insensitive towards application of the translation inhibitor cycloheximide prior to isolation of the membranes, implying the involvement of a regulatory enzyme in the electron transport in vivo.Abbreviations BPDS bathophenanthroline disulfonate - BTP 1,3-bis[tris(hydroxymethyl)methylamino]-propane - PM plasma membrane     

Sidedness of yeast plasma membrane vesicles and mechanisms of activation of the ATPase by detergents

The binding of concanavalin A and of fluorescein 5'-isothiocyanate indicate similar amount of right-side-out and inside-out vesicles in plasma membrane vesicles from either glucose-starved or glucose-fermenting yeast cells. These vesicles contain low-activity and high-activity states of the ATPase, respectively. Unmasking of latent active sites can explain the limited ATPase activation (about 2-fold) produced by several detergents on both kinds of vesicles. On the other hand, lysophosphatidic acid (oleoyl) produces a 7-fold activation of the ATPase in vesicles from glucose-starved cells. This effect is accompanied by a change in Km of the enzyme and probably reflects a direct action of the detergent on the ATPase. A similar activation and Km change can be obtained by sonication of the vesicles, although in this case soybean phospholipids are required for maximal activity. Apparently the low-activity state of the yeast plasma membrane ATPase can be activated not only by glucose metabolism 'in vivo' (mechanism unknown) but also by some detergents and physical treatments 'in vitro'. Experiments with purified ATPase from glucose-starved cells also indicate that lysophosphatidic acid (oleoyl) specifically activates the enzyme. These results suggest a note of caution on considering the usual interpretation of the effects of detergents on membrane enzymes, which only take into account the unmasking of latent active sites.     

Subunit specific antisera to the Escherichia coli ATP synthase: effects on ATPase activity, energy transduction, and enzyme assembly

We obtained antisera to each of the five subunits (α, β, γ, δ, and ?) of the F₁ portion of the proton-translocating ATPase from Escherichia coli (ECF₁). No cross-reaction between the antiserum to a given subunit and any of the other four subunits was observed by Ouchterlony immunodiffusion. The α antiserum reacted only with the denatured α chain. Antibodies to either subunit β or subunit γ inhibited the ATPase activity of the enzyme. The ATPase activity of the holoenzyme in the everted membrane vesicles was just as sensitive as purified ECF₁ to inhibition by the anti-β or anti-γ serum. A prolonged digestion of ECF₁ with trypsin removed intact γ from ECF₁, but did not alter the sensitivity of the ATPase to inhibition by the anti-γ serum. Proteolytic fragments were isolated from the trypsinized enzyme. They gave an immunoprecipitation band with the anti-γ serum, but none of the other subunit antisera. The antiδ serum detached ECF₁ from everted membrane vesicles and completely blocked both the ATP- and respiration-dependent pyridine nucleotide transhydrogenase, an energylinked membrane function. The δ antiserum had no effect on the ATPase activity of the ECF₁. The e antiserum stimulated the ATPase activity of purified ECF₁ as shown previously (P. P. Laget and J. B. Smith, Arch. Biochem. Biophys.197, 83, 1979), but strongly inhibited the holoenzyme in membrane vesicles. The α antiserum completely blocked the ATP-driven transhydrogenase. The same antiserum maximally inhibited the respiratory chain-driven reaction by only 35%. These observations indicate that the antiserum selectively affected energy transduction mediated by the ATPase. The protonmotive force generated by substrate oxidation was probably not dissipated by the ? antiserum. Adsorbing the δ or ? antiserum with everted membrane vesicles selectively removed those antibodies that reacted with membrane-bound ATPase. The adsorbed sera still reacted strongly with purified ECF₁, and prevented it from restoring ATP-dependent proton translocation in ECF₁-depleted vesicles. Therefore, it appears that more of the δ and the ? subunit is exposed in the purified ECF₁ molecule than in the membrane-bound enzyme.     

Proline transport activity in Escherichia coli membrane vesicles of different buoyant densities.

Cytoplasmic membrane vesicles prepared by lysis of Escherichia coli W 3110 spheroplasts in a French press at 0 degrees C are heterogeneous with respect to density due to membrane protein aggregation as a result of lateral phase separation of membrane phospholipids and to the presence of more or less outer membrane. These different vesicle classes can be separated on isopycnic density gradients. Assays for various membrane-associated functions show that the membranes differ not only with respect to density and structure but also with respect to function. The proline transport system (as detected by uptake experiments with the artificial electron donor ascorbate-phenazine methosulfate) shows maximal activities in membrane fractions that have considerably higher densities than the normal cytoplasmic membrane. This is always the case, whether vesicles are isolated from membranes that exhibit a temperature-induced protein aggregation or not. A correlation between high proline transport activity and the presence of vesicles with double membranes (consisting of outer and inner membrane) has been established. The possibility that the outer membrane protects the transport system in the cytoplasmic membrane during the isolation of vesicles is discussed.     

The DCCD-binding polypeptide is close to the F1 ATPase-binding site on the cytoplasmic surface of the cell membrane of Escherichia coli

Removal of the F₁ ATPase from membrane vesicles of $\text{Escherichia}\text{coli}$ resulted in leakage of protons across the membrane through the F_O portion of the ATPase complex. The leakage of protons was prevented by antiserum to the N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide in everted but not in “right-side out” membrane vesicles. The antiserum prevented the rebinding of F₁ ATPase to F₁-stripped everted membrane vesicles. It is concluded that in F₁-depleted vesicles the DCCD-binding polypeptide is exposed on the cytoplasmic surface of the cell membrane at or close to the binding site of the F₁ ATPase.     

Orientation of Membrane Vesicles from Escherichia coli as Detected by Freeze-Cleave Electron Microscopy

The application of freeze-cleave electron microscopy to whole cells of Escherichia coli revealed that the particles exposed on the resulting two inner membrane faces are asymmetrically distributed. This method can therefore be used to determine the orientation of membrane vesicles from E. coli. Membrane vesicles freshly prepared in potassium phosphate buffer (K(+)-vesicles) by osmotic lysis of spheroplasts consisted almost entirely of right-side-out vesicles. Their size suggested that each cell gives rise to one vesicle. When the membrane vesicles were subjected to one cycle of freezing and thawing, the number of inside-out vesicles rose to about 25%. However, due to the small size of most of the inside-out vesicles, these contribute only 2 to 3% of the total membrane surface area of the preparation. The inside-out vesicles appear to arise from infoldings of the membrane of right-side-out vesicles. They also accumulate within the latter, thus producing multivesicular membrane sacs. Na(+)-vesicles (vesicles prepared in sodium phosphate buffer) subjected to freezing and thawing appeared to lose structural rigidity more than did K(+)-vesicles. In contrast to the membrane vesicles prepared by the osmotic lysis of spheroplasts, those obtained by breaking intact cells by a single passage through a French pressure cell were uniformly very small (only 40 to 110 nm in diameter); approximately 60 to 80% were inside-out. To reconcile the polarity of the membrane vesicles with the enzymic activities of such preparations, we propose that "dislocation" of membrane proteins occurs during osmotic lysis of spheroplasts.     

Proline transport activity in Escherichia coli membrane vesicles of different buoyant densities

Cytoplasmic membrane vesicles prepared by lysis of Escherichia coli W 3110 spheroplasts in a French press at 0° C are heterogeneous with respect to density due to membrane protein aggregation as a result of lateral phase separation of membrane phospholipids and to the presence of more or less outer membrane. These different vesicle classes can be separated on isopycnic density gradients. Assays for various membrane-associated functions show that the membranes differ not only with respect to density and structure but also with respect to function.The proline transport system (as detected by uptake experiments with the artificial electron donor ascorbate-phenazine methosulfate) shows maximal activities in membrane fractions that have considerably higher densities than the normal cytoplasmic membrane. This is always the case, whether vesicles are isolated from membranes that exhibit a temperature-induced protein aggregation or not. A correlation between high proline transport activity and the presence of vesicles with double membranes (consisting of outer and inner membrane) has been established. The possibility that the outer membrane protects the transport system in the cytoplasmic membrane during the isolation of vesicles is discussed.     

The site of inhibition of photosynthetic electron transfer by amphotericin B.

The inhibitory effect of the polyene antibiotic, amphotericin B, on photosynthetic electron transfer has been investigated. Treatment of chloroplasts with the inhibitor results in the release of plastocyanin from its site in the chloroplast membrane. This release is accompanied by a shift in the pH curve for ferricyanide photoreduction from water, which is similar to that observed when chloroplasts are treated by sonication or passage through a French press. Delayed light emission from photosystem 2 is not destroyed by amphotericin B treatment, indicating that photosystem 2 is not damaged. Amphotericin B does not inhibit photoreduction of ferricyanide from water by chloroplast preparations which are deficient in plastocyanin, such as maize bundle-sheath chloroplast fragments, Euglena chloroplasts, or maize mesophyll chloroplasts passed through a French press. Chloroplasts treated with amphotericin B are not able to photooxidize plastocyanin. This result demonstrates that little structural damage occurs to the membrane during treatment with the antibiotic as a capacity to photooxidize plastocyanin is observed only in damaged chloroplast membranes.     

Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose: H+ carrier

The orientation of the lactose:H+ carrier of Escherichia coli in various preparations of native and reconstituted vesicles is determined with two impermeant, macromolecular probes: antibodies directed against the C-terminal decapeptide of the carrier and carboxypeptidase A (EC 3.4.17.1). Two methods are employed. Method I is based upon the digestion of all accessible and, therefore, presumably external, C termini of the carrier with carboxypeptidase A and detection of the remaining, internal C termini with 125I-labelled anti-(C-terminus) antibody after electrophoresis of the carrier in the presence of sodium dodecyl sulfate and transfer to nitrocellulose filters. Method II is based upon the binding of 125I-labelled anti-(C-terminus) antibody to the external C termini of the carrier in vesicles and the subsequent isolation of bound antibody by centrifugation. The labelled antibodies are calibrated using a preparation of inside-out vesicles prepared by high-pressure lysis of strain T206. The carrier content is determined by substrate binding. Because the C terminus of the carrier is known to reside on the cytoplasmic side of the membrane, these methods can also be used to determine the sidedness of various preparations of membrane vesicles. Spheroplasts are confirmed to contain carrier molecules of a single orientation, corresponding to that in right-side-out vesicles. In contrast, in purified cytoplasmic membrane vesicles and in crude membrane preparations obtained by sonication or by high-pressure lysis, 96% of the C termini are accessible to carboxypeptidase A, even after repeated sonication. This implies that nearly all carrier molecules in these preparations possess an orientation opposite to that in the cell or in right-side-out vesicles. In proteoliposomes containing carrier reconstituted or purified and reconstituted by two different methods, only 48% of the carrier molecules are oriented in the same way as in the cell. Subjecting such proteoliposomes to cycles of freezing and thawing or to sonication results in a reshuffling of carrier molecules between the inside-out and right-side-out populations while maintaining 41% in the right-side-out orientation. Digestion of the C terminus of the carrier with carboxypeptidase A does not alter either galactoside binding or countertransport. Thus carrier molecules of the inside-out orientation cannot be selectively inactivated. Additionally, an antiserum directed against the purified carrier is demonstrated to contain nearly exclusively anti-(C-terminus) antibodies, which can, in principle, be used in Method I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stereospecificity of NADH-ferricyanide reductase is a convenient marker for the endoplasmic reticulum of plant cells

The stereospecificity of NADH-ferricyanide reductase and NADH-cytochrome c reductase in the endoplasmic reticulum (ER) for the α-hydrogen on the nicotinamide ring is presented as a very sensitive and convenient assay to detect ER contamination in preparations of membranes lacking α-specific NADH-acceptor reductase, such as the plasma membrane and the tonoplast. The experimental details of the assay are given and the limitations explored (time-course, amount of protein, possible side reactions, speed, reproducibility, etc.). The NADH-ferricyanide reductase activity of plasma membranes from spinach and sugarbeet leaf was completely β-specific and always showed a latency (increase upon addition of Triton X-100), whereas the α-specificity in the ER was non-latent. This is consistent with the presence of mainly right-side-out vesicles in preparations of plasma membranes with the binding site for NADH and ferricyanide on the inner, cytoplasmic surface. In contrast, right-side-out ER vesicles have the binding site on the outer, cytoplasmic surface. The addition of as little as 1% of the α-specific ER (on an NADH-ferricyanide activity basis) to the spinach leaf plasma membrane could be detected with the stereospecificity assay. Wheat root plasma membrane showed some α-specificity (in addition to β-specificity) which was probably due to ER contamination since the activity was non-latent. The stereospecificity assay is also shown to be useful in monitoring the separation of tonoplast vesicles from ER vesicles by countercurrent distribution of a light microsomal fraction. It follows that the NADH-acceptor reductase activities in preparations of plasma membrane and tonoplast are due to distinct enzymes characteristic for those membranes.     

Immunoferritin labeling of respiratory nitrate reductase in membrane vesicles of Bacillus licheniformis and Klebsiella aerogenes

The indirect immunoferritin labeling method was used to localize the membrane-bound respiratory nitrate reductase in membrane vesicles and protoplasts or spheroplasts of Bacillus licheniformis and Klebsiella aerogenes, respectively. For a comparison of the labeling of the various vesicle preparations, which differed not only in size but also in the percentage of inside-out orientation, a quantification of the results was needed to circumvent the problem of non-specifically bound ferritin. From the results the sidedness of the nitrate reductase in the cytoplasmic membrane of the abovementioned bacteria was determined as being cytoplasmic in B. licheniformis and as transmembranous in K. aerogenes.Non-Standard Abbreviations PBS phosphate buffered saline - IgG immunoglobulin G     

Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.     

Comparative inhibition studies of the phosphotransferase and glycerophosphate acylation systems in membrane vesicles of Escherichia coli

Purified membrane vesicles were treated with various reagents specific for different amino acid side-chains. Titration of sulfhydryl groups with specific reagents shows that the sulfhydryl content of membrane vesicles as estimated directly is similar to that found by treating spheroplasts or cells and then isolating the membrane vesicles. The blocking of sulfhydryl groups specifically inhibits the α-methylglucoside transport system (phosphotransferase system), whereas the glycerophosphate acylation system is not affected. The kinetics of inhibition of the first system show that a high reactivity of the sulfhydryl groups is involved. Inhibition of the acyltransferase activity by sulfhydryl reagents occurs only on partial denaturation of the membranes induced by mild sonication, heat or toluene treatment. The Inhibition is at the level of the glycerol 3-phosphate:acyl thioester acyltransferase.The effects of sonication and/or sulfhydryl reagents were measured by sulfhydryl titration, by assays of NADH oxidase and d-lactate dehydrogenase activities, as well as by 1-anilino-8-naphthalene sulfonate binding. The results support the hypothesis that the acyltransferase system is embedded within the membrane and that the readily accessible permease system is closer to (or at) the surface of the membrane.     

Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

Abstract Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.