Isolation of a sealed homogeneous population of inner membrane fragments with inverted orientation from rat liver mitochondria using specific lectin immunoprecipitation

A novel method for the isolation of well-defined populations of inside-out vesicles from rat liver mitochondria is described. The technique utilizes specific immunoprecipitation of vesicles with accessible carbohydrate residues from a mixed population of inner membrane fragments using wheat germ agglutinin and anti-wheat germ agglutinin IgG. The unprecipitated fraction comprises 30--50% of the original population and exhibits little or no cytochrome c oxidase activity as estimated with exogenous cytochrome c as substrate. Addition of deoxycholate to promote membrane disruption results in an 8--10-fold increase in enzymic activity compared to only 1.5--2.0-fold stimulation in standard preparations of submitochondrial particles. It is concluded that the lectin affinity-purified membranes represent a sealed homogeneous (90--95% pure) population of inside-out inner membrane vesicles.     

Flow cytometry of spinach chloroplasts : determination of intactness and lectin-binding properties of the envelope and the thylakoid membranes

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes.     

Association of gangliosides with the lymphocyte plasma membrane studied using radiolabels and spin labels

Gangliosides are known to act as potent suppressors of lectin-stimulated lymphocyte activation when added to the culture medium. Since this effect may be mediated via ganglioside association with (or insertion into) the plasma membrane, we have used 3H- and spin-labelled derivatives of mixed gangliosides to probe the nature of this interaction. Gangliosides bind rapidly to the lymphocyte membrane and show no preference for association with either inside-out or right-side-out membrane vesicles. Around 20% of the bound gangliosides can be removed by repetitive washing, and a further 22-28% by treatment with pronase for 1 h, suggesting that this fraction is tightly bound to membrane proteins at the cell surface. The ESR spectrum of membrane-bound gangliosides did not resemble the spin-exchanged spectrum of micellar spin-labelled gangliosides in aqueous solution, but was similar to that seen for 5 mol% ganglioside spin label in liposomes of egg phosphatidylcholine. This suggests that the bulk of the membrane-bound gangliosides are inserted and molecular dispersed in the lymphocyte membrane. Binding of wheat-germ agglutinin to lymphocyte-associated gangliosides results in specific immobilization of the carbohydrate headgroup, while concanavalin A and other lectins have little or no effect on oligosaccharide mobility. Membrane-inserted gangliosides show a response to lectin binding which is qualitatively different from that seen for gangliosides in bilayers of phosphatidylcholine.     

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.     

Further characterization of the outer membrane ofSerratia marcescens and an oxacilline-sensitive mutant: Surface carbohydrates and outer membrane lipid composition

Whole cells and isolated outer membranes ofSerratia marcescens and an oxacilline-sensitive mutant could be agglutinated only with concanavalin A and wheat germ agglutinin. Different amounts of carbohydrates were accessible to the lectins in the two strains, and different amounts of carbohydrate seemed to be exposed at the outer cell surface and the inner side of the outer membrane. The fatty acid composition of the isolated outer membrane revealed small but significant quantitative difference. The increased sensitivity towards various antibacterial agents was thought to be the result of an altered hydrophobic-hydrophilic interaction of the outer membrane components.     

Sidedness of plant plasma membrane vesicles altered by conditions of preparation

Right-side-out vesicles of plasma membrane from soybean (Glycine max Merr.) were isolated by aqueous two-phase partition. Inside-out vesicles were formed when these preparations were diluted or frozen and thawed. Sidedness (orientation) was determined by preparative free-flow electrophoresis, concanavalin A binding, and ATPase latency. Under usual conditions of aqueous two-phase partition, the bulk of the vesicles were strongly reactive with concanavalin A-peroxidase and showed a high level of structure-linked latency as expected of a right-side-out (cytoplasmic-side-in) orientation. The vesicles migrated as a single electrophoretic peak. When frozen and thawed, vesicle diameters were reduced and a second population of vesicles of increased electrophoretic mobility was obtained. This second population of vesicles was weakly reactive with concanavalin A-peroxidase and showed low latency as expected of an inside-out (cytoplasmic-side-out) orientation. If the plasma membrane vesicles were diluted with water, a mixture of right-side-out and inside-out vesicles again was obtained. However, some of the cytoplasmic-side-out vesicles that were concanavalin A-unreactive and had low ATPase latency migrated more slowly as a second, less electronegative peak, upon free-flow electrophoresis. The results suggest that right-side-out and inside-out plasma membrane vesicles differ in electrophoretic mobility but that both the orientation and the absolute electrophoretic mobility of the differently oriented vesicles may be influenced by the preparative conditions.     

The preparation of rat liver lysosome membranes. Several membrane proteins contain complex oligosaccharides

Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze-thaw shock and membranes were isolated. The release of beta-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.     

Characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry

The isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes were characterized by lectin cytochemistry using concanavalin A (ConA), Ricinus communis agglutinin 120 (RCA-120), and wheat germ agglutinin (WGA). We found that RCA-120, ConA, and WGA bind to these membranes. The distribution of the lectins on the isolation membranes was heterogeneous, mainly found on the rims, which we referred to as the peripheral dilated portion. When the rims fused and thus formed autophagosomes the apparent sites of fusion were strongly labeled by the lectins. After autophagosomes were transformed to autolysosomes by fusion with lysosomes, the limiting membranes became more densely and homogeneously labeled with the lectins. We previously reported that cytochrome P-450 does not exist on the limiting membranes of the autophagosomes. Taken together, these results suggest that the isolation membranes may originate not from endoplasmic reticulum membranes but from some post-Golgi membranes that contain complex type N and/or O-linked oligosaccharide chains.     

Chemically-induced cation permeability in red cell membrane vesicles. The sidedness of the response and the proteins involved.

Cation fluxes were measured in right-side-out and inside-out vesicles obtained from human red cells. Rubidium, which is spontaneously released at very slow rates, can be rapidly released from both types of vesicle by addition of valinomycin. P-Chloromercuriphenyl sulfonic acid (PCMBS) also increases the cation permeability of the vesicles with reversal to normal after addition of dithiothreitol. The effect of PCMBS is considerably larger and appears faster in the inside-out vesicles as compared to the right-side-out vesicles, the difference being greater at low temperatures. These data indicate that the SH groups responsible for the changes in cation permeability are more accessible from the inside face of the membrane. The response to PCMBS was not diminished after selective removal of extrinsic proteins by alkaline extraction, and/or after the membranes were exposed to proteolytic enzymes. The major polypeptide component remaining in vesicles after both treatments was a 17 000-dalton transmembrane fragment derived from band 3 which might, therefore, be responsible for the permeability response. Addition of Ca2+ to either right-side-out or inside-out vesicles, in the presence or absence of ionophore A23187, was without effect on monovalent cation permeability, indicating that the mechanism of Ca2+-induced K+ permeation was lost or inactivated during the preparation of the vesicles.     

Isolation and characterization of a plasma membrane fraction from sea urchin sperm exhibiting species specific recognition of the egg surface

A method is described for isolating preparative quantities of plasma membranes from sea urchin sperm. The final membrane fraction is homogeneous by sucrose density sedimentation and is enriched in adenylate cyclase as well as in the four glycoproteins accessible to radioiodination of intact sperm. The electrophoretic profiles of sperm membranes from three sea urchin species are very similar. The membrane preparation consists primarily of sealed vesicles which release carboxyfluorescein when exposed to detergents or distilled water. Ninety-two percent of the 125I-labeled vesicle material binds to wheat germ lectin columns, suggesting a right-side-out orientation. The isolated sperm membrane vesicles exhibit species specific adhesion to the surfaces of sea urchin eggs; this adhesion is blocked by pretreatment of the vesicles with trypsin or egg jelly. This method will be useful for isolating biologically active sperm membrane components involved in sperm-egg recognition during fertilization.     

The toolbox of vesicle sidedness determination

Vesicles prepared from cellular plasma membranes are widely used in science for different purposes. The outer membrane leaflet differs from the inner membrane leaflet of the vesicle, and during vesicle preparation procedures two types of vesicles will be generated: right-side-out vesicles, of which the outer leaflet is topologically equivalent to the outer monolayer of the cellular plasma membrane, and inside-out vesicles. Because two populations of vesicles exist, sidedness information of the vesicle preparation is indispensable. This note focuses on the ins and outs of sidedness determination of vesicles and compares various methodologies used to establish this ratio.     

Calcium binding to extracellular sites of skeletal muscle calcium channels regulates dihydropyridine binding

The binding of dihydropyridine (PN200-110) to skeletal muscle microsomes (which were 84% sealed inside-out vesicles) was not influenced by the addition of calcium or magnesium nor by addition of their chelators (EDTA or EGTA) unless the vesicles were pretreated with the calcium-magnesium ionophore A23187 and EDTA to remove entrapped cations. Separation of inside-out vesicles from right-side-out vesicles by wheat germ agglutinin chromatography revealed that only the right-side-out vesicles exhibited a calcium-, magnesium-, and chelator-dependent binding of PN200-110. Dihydropyridine binding to cardiac sarcolemma membranes (which were 46% inside-out) and to solubilized skeletal muscle membranes was inhibited by EDTA and could be fully restored by 10 microM calcium or 1 mM magnesium. Calcium increased PN200-110 binding to partially purified rabbit skeletal muscle calcium channels from 3.9 pmol/mg protein to 25.5 pmol/mg protein with a pK0.5 = 6.57 +/- 0.059 and a Hill coefficient of 0.56 +/- 0.04. Magnesium increased binding from 0.7 pmol/mg protein to 16.8 pmol/mg protein with a pK0.5 = 3.88 +/- 0.085 and a Hill coefficient of 0.68 +/- 0.074. These studies suggest that calcium binding to high affinity sites or magnesium binding to low affinity sites on the extracellular side of skeletal muscle T-tubule calcium channels regulates dihydropyridine binding. Further, similar calcium and magnesium binding sites exist on the cardiac calcium channel and serve to allosterically regulate dihydropyridine binding.     

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.     

Nature of the lectin-induced activation of plasma membrane Mg2+ATPase.

The Mg2+ATPase activity of liver plasma membranes decreases markedly with increasing temperature above 30 degrees. This negative temperature dependency is counteracted by the binding of wheat germ agglutinin, concanavalin A, or Ricinus communis agglutinin (at concentrations greater than or equal 0.5 mg/ml) to membranes prior to assay of the enzyme. With one of these lectins bound, the enzyme has a single energy of activation between 20 degrees and 45 degrees. The binding of dimeric succinyl concanavalin A, soybean agglutinin, fucose-binding lectin from Lotus tetragonolobus, or the leucoagglutinin from Phaseolus vulgaris does not alter the temperature dependency of the enzyme. The latter two lectins, however, do prevent the concanavalin A-induced activation of the enzyme at 37 degrees. At saturating substrate concentrations, the enzyme is not inhibited by any of the lectins tested over a wide range of concentrations. Cytochalasin B and colchicine separately or in combination have little influence on the lectin-induced enhancement of enzyme activity. Chlorpromazine and vinblastine sulfate each partially prevent the activation and in combination do so completely. Treatment of the membranes with the detergent Lubrol-PX or phospholipase A prevents activation of the enzyme by concanavalin A. The results are consistent with a restriction by the lectin of an environment which is normally too disordered for maximal enzyme activity above 30 degrees.     

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)     

Subfractionation of cardiac sarcolemma with wheat-germ agglutinin.

The properties of highly purified bovine cardiac sarcolemma subfractionated with the lectin, wheat-germ agglutinin (WGA) were studied. Two different membrane subfractions were isolated, one which was agglutinated in the presence of 1.0 mg of WGA/mg of protein (WGA+ vesicles) and a second fraction which failed to agglutinate (WGA- vesicles). These two membrane fractions had quantitatively different rates of Na+/K+-dependent, ouabain-sensitive ATPase and Na+/Ca2+ exchange activities, yet a similar protein composition, which suggests that they were both derived from the plasma membrane. WGA- vesicles had a decreased number of [3H]quinuclidinyl benzilate-binding sites and no detectable [3H]nitrendipine-binding sites. Electron-microscopic and freeze-fracture analysis showed that the WGA+ fraction was composed of typical spherical sarcolemmal vesicles, whereas the WGA- fraction primarily contained elongated tubular structures suggestive of the T-tubule vesicles which were previously isolated from skeletal muscle. Assays of marker enzymes revealed that these fractions were neither sarcoplasmic reticulum nor plasma membrane from endothelial cells. Moreover, WGA agglutination did not result in the separation of right-side-out and inside-out vesicles. On the basis of these findings we propose that the WGA+ fraction corresponds to highly purified sarcolemma, whereas the WGA- fraction may be derived from T-tubule membranes.     

Preparation and characterization of unilamellar myelin vesicles

Myelin vesicles have been obtained from isolated rat brain myelin and shown by electron microscopy to consist of single bilayer membranes. The yield of the preparation is approximately 25% of the myelin proteins. The vesicles show a typical myelin protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contain activity for the myelin marker enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase). The preparation consists of both inside-out and right-side-out vesicles, and the proportion in each orientation varies from one preparation to another. The occurrence of two populations is demonstrated by the observation that hypotonically lysed vesicles compete to a greater extent than intact vesicles in a competitive enzyme-linked immunosorbent assay with myelin basic protein antiserum. In addition, only a portion of the CNPase activity of the vesicles is trypsin-sensitive and detectable in the absence of detergent; the remaining, trypsin-insensitive activity is present in detergent-disrupted membranes. Thus, there are vesicle populations in which myelin basic protein and CNPase are accessible and others in which they are inaccessible. A population of uniformly oriented right-side-out vesicles has been obtained by ConA-Agarose affinity column chromatography and elution of the bound fraction with methyl-alpha-D-mannopyranoside. In the absence of detergent, less than 10% of the total CNPase activity of these vesicles can be demonstrated, suggesting that the active site of CNPase is opposite to that of the ConA binding site and, therefore, appears to be on the cytoplasmic face of the myelin membrane.     

Characterization of the Carbohydrate Chain on the Substance P Receptor in the Rat Brain Cortex: Effect of Lectins on [3H]Substance P Binding

The characteristics of the carbohydrate chain on the rat cerebral cortical substance P (SP) receptor were studied. We examined the effects of pretreatment with three lectins (concanavalin A, wheat germ agglutinin, lens culinaris agglutinin) on the [3H]SP binding activities. Each lectin can bind to the specific carbohydrate chain. Among these lectins, only concanavalin A inhibited specific [3H]SP binding by reducing the affinity of the binding sites. The inhibitory action of concanavalin A was dose-dependent and diminished by the addition of alpha-methyl-D-mannoside. The present results suggest that the rat cortical SP receptor has either a biantennary complex-type or a high mannose-type of carbohydrate chain, and that the carbohydrate chain is implicated in the SP binding activity of the SP receptor system.     

Orientation of vesicles isolated from baso-lataral membranes of renal cortex

Baso-lateral membranes were isolated from the canine and porcine kidney cortex by several different methods currently in use. Sidedness of the isolated membrane vesicles was determined by procedures using 1. ouabain-sensitive (Na+K+)ATPase assays in the presence and in the absence of sodium dodecylsulfate or digitoxigenin plus monensin, 2. (Na+, K+, Mg2+)ATPase assays with valinomycin, 3. sialidase accessibility, and 4. binding of hydrophilic and lipophilic cardiac glycosides. The (Na+K+)ATPase activity in the membrane preparation was increased 10-fold of that found in the crude homogenate. Isolated membrane vesicles, prepared by different techniques, were all found to be overwhelmingly of right-side-out orientation;namely, right-side-out = 51-68%, inside-out = 4-13%, and unsealed vesicles = 26-42%. Results of sidedness determinations by different methods showed a good agreement. Thus, predominantly right-side-out oriented vesicles are formed during conventional isolation procedures for membranes of the kidney cortex.     

Reconstitution of Escherichia coli membrane vesicles with D-amino acid dehydrogenase

When purified D-amino acid dehydrogenase [Olsiewski, P. J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 255, 4487] is incubated with right-side-out membrane vesicles from Escherichia coli, the enzyme binds to the membrane in a time- and concentration-dependent manner. As a result, the vesicles acquire the ability to oxidize D-alanine and catalyze D-alanine-dependent active transport. Similarly, incubation of D-amino acid dehydrogenase with inside-out vesicles results in binding of enzyme and D-alanine oxidase activity. Antibody inhibition studies indicate that the enzyme is bound exclusively to the inner cytoplasmic surface of the membrane in native vesicles (i.e., membrane vesicles prepared from cells induced for D-amino acid dehydrogenase). In contrast, similar studies with reconstituted vesicles demonstrate that enzyme binds to the surface exposed to the medium regardless of the orientation of the membrane. Thus, enzyme bound to right-side-out vesicles is located on the opposite side of the membrane from where it is normally found. Remarkably, in the presence of D-alanine, reconstituted right-side-out and inside-out vesicles generate electrochemical proton gradients of similar magnitude but opposite polarity, indicating that enzyme bound to either surface of the membrane is physiologically functional. The results suggest that vectorial proton translocation via the respiratory chain occurs at a point distal to the site where electrons enter the respiratory chain from the primary dehydrogenase, a conclusion that is inconsistent with the notion that the dehydrogenase forms part of a proton-translocating loop.