A micro-batchwise technique method for rapid reconstitution of functionally active mitochondrial ADP/ATP carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers

A method for rapid reconstitution of ADP/ATP carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers mitochondria in proteoliposomes is described. The method is based on the well known property of the Amberlite resin to absorb the detergent allowing proteoliposome formation. This has been achieved by a micro-batchwise technique, using a rotating plate stirrer. An evaluation of the optimal conditions, in comparison with the more usual column method is presented. The purified ADP/ATP carrier, incorporated in proteoliposomes by this method, shows a high transport activity and a higher specific activity with respect to proteoliposomes obtained by the column procedure. Furthermore the proteoliposomal preparations are more homogeneous in size, with a diameter ranging from 300 to 350 nm. The method is suitable for the reconstitution of other membrane transport proteins.     

Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associated with planar azolectin membrane, proteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgCl electrodes and voltmeter. The direction of the electric field in proteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

Membrane protein reconstitution for functional and structural studies

Membrane proteins are involved in various critical biological processes,and studying membrane proteins represents a major challenge in protein biochemistry.As shown by both structural and functional studies,the membrane environment plays an essential role for membrane proteins.In vitro studies are reliant on the successful reconstitution of membrane proteins.This review describes the interaction between detergents and lipids that aids the understanding of the reconstitution processes.Then the techniques of detergent removal and a few useful techniques to refine the formed proteoliposomes are reviewed.Finally the applications of reconstitution techniques to study membrane proteins involved in Ca2+ signaling are summarized.     

A new method for the reconstitution of membrane proteins into giant unilamellar vesicles

In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.     

Functional reconstitution of prokaryote and eukaryote membrane proteins

A new strategy for the functional reconstitution of membrane proteins is described. This approach introduces a new class of protein stabilizing agents--osmolytes--whose presence at high concentration (10-20%) during detergent solubilization prevents the inactivations that normally occur when proteins are extracted from natural membranes. Osmolytes that act in this way include compounds such as glycerol and higher polyols (erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and certain amino acids (glycine, proline, betaine). The beneficial effects of osmolytes are documented by reconstitution of a variety of prokaryote and eukaryote membrane proteins, including several proton- and calcium-motive ATPases, cation- and anion-linked solute carriers (symport and antiport), and a membrane-bound hydrolase from endoplasmic reticulum. In all cases, the presence of 20% glycerol or other osmolyte during detergent solubilization led to 10-fold or more increased specific activity in proteoliposomes. These positive effects did not depend on use of any specific detergent for protein solubilization, nor on any particular method of reconstitution, but for convenience most of the work reported here has used octylglucoside as the solubilizing agent, followed by detergent-dilution to form proteoliposomes. The overall approach outlined by these experiments is simple and flexible. It is now feasible to use reconstitution as an analytical tool to study the biochemical and physiological properties of membrane proteins.     

Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes.

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associtated with planas azolectin membrane, ptoteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgC1 electrodes and voltmeter. The direction of the electric field inproteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.     

A Simple Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.     

Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities.

Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP-xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent-solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters.     

Fast, efficient reconstitution of the cyclooxygenases into proteoliposomes

To study the physical and catalytic properties of purified membrane proteins, it is often necessary to reconstitute them into lipid bilayers. Here, we describe a fast efficient method for the direct incorporation of cyclooxygenase-1 and -2 (COX-1 and -2) isozymes into liposomes without loss of activity. Purified COX-1 and -2 spontaneously incorporate into large unilamellar vesicles produced from a mixture of DOPC:DOPS (7:3) that has been doped with oleic acid. When incorporation was measured by comparing cyclooxygenase activity to total phospholipid in the proteoliposomes, molar reconstitution ratios of 1000:1 (phospholipid:COX) were obtained. Electron paramagnetic resonance spectroscopic spin counting analysis of proteoliposomes formed with nitroxide spin-labeled COX-2 gave a nearly identical phospholipid:COX ratio, confirming that incorporation had no effect on enzyme activity, and demonstrating that the efficiency of protein incorporation is sufficient for EPR spectroscopic analysis. The spontaneous incorporation of cyclooxygenase into intact liposomes allows only insertion into the outer leaflet for this monotopic enzyme, an orientation confirmed by immunogold staining of the proteoliposomes. This method of reconstitution into liposomes may be generally applicable to the class of monotopic integral membrane proteins typified by the cyclooxygenase isozymes.     

Lactose-proton symport by purified lac carrier protein

The lac carrier protein of Escherichia coli was purified by an improved procedure and its activity assayed by a rapid filter method. Following reconstitution of the carrier by octyl glucoside dilution, proteoliposomes were concentrated by filtration on a microporous filter. Lactose accumulation by adsorbed or entrapped proteoliposomes is driven by an artificially imposed pH gradient (interior alkaline), by a membrane potential (interior negative), or by a combination of both forces. Activity is almost completely abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone or by the competitive inhibitor thiodigalactoside. Addition of lactose to proteoliposomes under appropriate conditions results in alkalinization of the external medium. This effect is not observed with liposomes devoid of lac carrier or in the presence of proton conducting agents. The results provide a strong indication that the lac gamma gene product is the only protein in the cytoplasmic membrane of Escherichia coli required for lactose-proton symport.     

Reconstitution of transport function of vacuolar H(+)-translocating inorganic pyrophosphatase.

A procedure for reconstitution of the transport function of the vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1) prepared from etiolated hypocotyls of Vigna radiata (mung bean) is described. The method entails sequential extraction of isolated vacuolar membrane (tonoplast) vesicles with deoxycholate and CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), combination of CHAPS-solubilized protein with phospholipid-cholesterol mixtures, dialysis, and glycerol density gradient centrifugation. The final proteoliposome preparation is 9-fold enriched for PPase activity and active in pyrophosphate (PPi)-energized electrogenic H(+)-translocation. Since both PPi hydrolysis and PPi-dependent H(+)-translocation by the proteoliposomes are indistinguishable from the corresponding activities of native tonoplast vesicles, the functional integrity of the H(+)-PPase appears to be conserved during solubilization and reconstitution. The high transport capacity and amenability of the reconstituted enzyme to both radiometric membrane filtration and fluorimetric H(+)-translocation assays, on the other hand, demonstrate its applicability to a broad range of transport studies. SDS-polyacrylamide gel electrophoresis of the proteoliposomes reveals selective enrichment of the M(r) 66,000, substrate-binding subunit of the H(+)-PPase and two additional polypeptides of M(r) 21,000 and 20,000. Although the M(r) 21,000 and 20,000 polypeptides have not been described previously, all attempts to reconstitute H(+)-PPase lacking these components were unsuccessful. It is therefore tentatively proposed that the M(r) 21,000 and 20,000 polypeptides, as well as the M(r) 66,000 subunit, are required for the productive reconstitution of PPi-dependent H(+)-translocation.     

Solubilization and reconstitution of hepatic System A-mediated amino acid transport. Preparation of proteoliposomes containing glucagon-stimulated transport activity

System A-mediated amino acid transport activity from rat liver plasma membrane vesicles has been solubilized and reconstituted into proteoliposomes using a freeze-thaw-dilution technique. The presence of cholate, at a cholate to protein ratio of 1:1, during the freeze-thaw step resulted in an enhancement in recoverable transport activity. The carrier required both phosphatidylcholine and phosphatidylethanolamine for optimal activity, but the addition of cholesterol to the reconstitution procedure appeared to have no significant effect on the resulting activity. A lipid to protein ratio of 20:1 yielded maximal transport activity. Sonication of the proteoliposomes provided some improvement in the accuracy of replicate assays for a given proteoliposome preparation. Isolated liver plasma membrane vesicles prepared from rats treated in vivo with glucagon in combination with dexamethasone contained stimulated System A activity. This enhanced transport activity could be solubilized and recovered in proteoliposomes generated from these plasma membranes. The data support the proposal that hormone regulation of the hepatic System A gene results in the de novo synthesis and plasma membrane insertion of the carrier protein itself.     

Reconstitution and partial purification of an amiloride-sensitive, cation channel from rabbit kidney

The aim of the present study was to reconstitute and purify an epithelial potassium channel from rabbit kidney. Renal brush border membrane vesicles (BBMV) were found to contain a potassium conductance which was inhibited by amiloride, 5-(N-methyl-N-isobutyl)amiloride (MIA) and by barium. Membrane vesicle proteins were solubilized and reconstituted in proteoliposomes. Channel activity was assayed using Acridine orange and the voltage sensitive dye, 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Both methods yielded similar results which indicated the presence of an amiloride-sensitive, cation channel in the proteoliposomes. This channel was more permeable to K than to Na and its activity was increased in reconstituted proteoliposomes as compared to native brush border membranes. We conclude that rabbit BBMV possess an amiloride sensitive cation channel. Channel activity was successfully reconstituted in proteoliposomes and the protein was partially purified during reconstitution.     

Nystatin-induced increase in photocurrent in the system ‘bacteriorhodopsin proteoliposome/bilayer planar membrane’

Proteoliposomes were reconstituted from bacteriorhodopsin sheets, asolectin and cholesterol with or without nystatin. Bacteriorhodopsin-mediated electrogenesis was monitored using (1) a proteoliposome suspension and phenyldicarbaundecaborane (PCB^?) probe or (2) proteoliposomes associated with planar bilayer membrane and orthodox electrometer techniques. In the light, PCB^? was shown to be taken up by proteoliposomes. The PCB^? uptake was inhibited by addition of nystatin to an incubation mixture with proteoliposomes if they were reconstituted in the presence of nystatin. Extraproteoliposomal nystatin was without influence if nystatin was omitted from the reconstitution mixture. The nystatin-containing proteoliposomes were associated with a planar bilayer asolectin membrane in the presence of Ca²⁺. It was found that in such a system, bacteriorhodopsin generated a photocurrent charging the proteoliposome-containing (cis-side) compartment negatively and the trans-side compartment positively. The photoresponse was shown to be increased several-fold by addition of nystatin to the trans-side solution. Nystatin addition was ineffective if proteoliposomes were reconstituted without nystatin. Taking into account that nystatin forms ion-permeable pores in a membrane only if present on both sides of the membrane and that this membrane is bilayer, one can explain the above data assuming that (1) the intraproteoliposomal solution does not mix with the extraproteoliposomal one when proteoliposomes are attached to a planar black membrane and (2) the attached proteoliposomes are separated from the trans-side bathing solution by a bimolecular membrane. If this is the case, nystatin in the trans-side bathing solution and inside the attached proteoliposome can form pores across that part of the planar membrane which separates the proteoliposome interior from the trans-side solution. Through these pores, H⁺ (pumped by bacteriorhodopsin from the cis-side solution into the proteoliposome interior) or some other intraproteoliposomal ions can be equilibrated with those in the trans-side solution. As a result, the bacteriorhodopsin-generated photocurrent increases.     

SNARE-mediated lipid mixing depends on the physical state of the vesicles

Reconstitution experiments have suggested that N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins constitute a minimal membrane fusion machinery but have yielded contradictory results, and it is unclear whether the mechanism of membrane merger is related to the stalk mechanism that underlies physiological membrane fusion. Here we show that reconstitution of solubilized neuronal SNAREs into preformed 100 nm liposomes (direct method) yields proteoliposomes with more homogeneous sizes and protein densities than the standard reconstitution method involving detergent cosolubilization of proteins and lipids. Standard reconstitutions yield slow but efficient lipid mixing at high protein densities and variable amounts of lipid mixing at moderate protein densities. However, the larger, more homogenous proteoliposomes prepared by the direct method yield almost no lipid mixing at moderate protein densities. These results suggest that the lipid mixing observed for standard reconstitutions is dominated by the physical state of the membrane, perhaps due to populations of small vesicles (or micelles) with high protein densities and curvature stress created upon reconstitution. Accordingly, changing membrane spontaneous curvature by adding lysophospholipids inhibits the lipid mixing observed for standard reconstitutions. Our data indicate that the lipid mixing caused by high SNARE densities and/or curvature stress occurs by a stalk mechanism resembling the mechanism of fusion between biological membranes, but the neuronal SNAREs are largely unable to induce lipid mixing at physiological protein densities and limited curvature stress.     

Measurement of proton pump activity of the thermophilic bacterium PS3 and Nitrobacter agilis at the cytochrome oxidase level using total membrane and heptyl thioglucoside

It is possible to prepare liposomal vesicles by solubilization of total bacterial membranes with n-heptyl beta-D-thioglucoside followed by reconstitution into proteoliposomes by a freeze-thaw-sonication procedure with soybean phospholipids. The resulting proteoliposomes from total membrane fraction of sufficiently aerated cells of the thermophilic bacterium PS3 containing cytochrome aa3 showed a reasonable H+ pumping activity upon addition of reduced cytochrome c. On the other hand, the proteoliposomes reconstituted from air-limited PS3 cells containing cytochrome o and those from Nitrobacter agilis cells containing cytochrome aa3 did not show H+ pumping upon addition of reduced cytochrome c, although the vesicles showed "respiratory control"; 3-4-fold stimulation of oxygen consumption took place upon addition of an uncoupler. In proteoliposomes prepared from PS3 membranes by this method, H+-translocating ATPase (F0 X F1) was successfully reconstituted as well, suggesting that this method has wide applicability for investigation of enzymes catalyzing transmembrane processes.     

Anemone toxin II receptor site of the lobster nerve sodium channel. Studies in membrane vesicles and in proteoliposomes

The receptor-site for the sea anemone toxin II from Anemonia sulcata (ATX) and its functional relationship with the Na+ channel were studied in plasma membrane preparations from lobster walking leg nerves. The modification of the 22Na influx by ATX was determined in membrane vesicles and in proteoliposomes prepared by reconstitution of detergent-extracted, unfractionated membrane particles into soybean liposomes. The effects of two other toxins, veratridine (VER) and tetrodotoxin (TTX), which bind to Na+ channel receptor-sites other than that for polypeptide toxins, were also studied, ATX and VER stimulated 22Na flux into membrane vesicles with K0.5 values in the order of 10(-7) and 10(-5) M, respectively. Positive cooperativity among these toxins was also seen; ATX displaces the K0.5 for VER towards lower VER concentrations. TTX abolishes the 22Na influx increment caused by ATX and/or VER with a K0.5 in the order of 10(-8) M. In proteoliposomes, in contrast, ATX modified the 22Na influx only at high concentrations (greater than 1 microM) and in the presence of VER. VER stimulation and TTX inhibition of the VER and the VER plus ATX modified fluxes, had the same characteristics as in the vesicle preparations. Measurable ATX and VER toxin effects were only seen in the presence of an outwardly directed K+ gradient for both vesicles and proteoliposomes. Detergent treatment and the reconstitution procedure seem to affect the functional properties of the ATX receptor site whereas the VER and the TTX sites remain unaltered.     

Preparation of reconstituted acetylcholine receptor membranes suitable for AFM imaging of lipid–protein interactions

The nicotinic acetylcholine receptor (nAChR) has been reconstituted in POPC vesicles at high lipid–protein (L/P) ratios for the preparation of supported lipid bilayers with a low protein density for studies of protein–lipid interactions using atomic force microscopy (AFM). Initial reconstitutions using a standard dialysis method with bulk L/P ratios ranging from 20:1 to 100:1 (w/w) gave heterogeneous samples that contained both empty vesicles and proteoliposomes with a range of L/P ratios. This is problematic because empty vesicles adsorb and rupture to form bilayer patches more rapidly than do protein-rich vesicles, resulting in the loss of protein during sample washing. Although it was not possible to find reconstitution conditions that gave homogeneous populations of vesicles with high L/P ratios, an additional freeze–thaw cycle immediately after dialysis did reproducibly yield a fraction of proteoliposomes with L/P ratios above 100:1. These proteoliposomes were separated by sucrose gradient centrifugation and used to prepare supported bilayers with well-separated individual receptors and minimal adsorbed proteoliposomes. AFM images of such samples showed many small features protruding from the bilayer surface. These features range in height from 1 to 5 nm, consistent with the smaller intracellular domain of the protein exposed, and have lateral dimensions consistent with an individual receptor. Some bilayers with reconstituted protein also had a small fraction of higher features that are assigned to nAChR with the larger extracellular domain exposed and showed evidence for aggregation to give dimers or small oligomers. This work demonstrates the importance of using highly purified reconstituted membranes with uniform lipid–protein ratios for AFM studies of integral membrane protein–lipid interactions.     

Solubilization and Reconstitution of the Mg2+/2H+ Antiporter of the Lutoid Tonoplast from Hevea brasiliensis Latex

The Mg2+/2H+ antiporter recently described on lutoid membrane (Z. Amalou, R. Gibrat, C. Brugidou, P. Trouslot, J.d'Auzac [1992] Plant Physiol 100: 255-260) was solubilized by octylglucoside and reconstituted into soybean liposomes using the detergent dilution method. Magnesium efflux or influx experiments were used to generate a H+ influx or efflux, respectively, monitored with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine. Both experiments gave saturable H+ fluxes as a function of internal or external Mg2+ concentrations with similar kinetic parameters Km and Vmax. The Km value for Mg2+ (about 2 mM) was identical to that previously found in lyophilized-resuspended lutoid (reference therein), whereas the Vmax value was 14-fold higher. Since only 10% of the initial proteins were recovered in proteoliposomes, and electrophoretic patterns of the two kinds of vesicles differed significantly, it was inferred that the increase in Vmax was due essentially to an enrichment of the protein antiporter in the reconstituted fraction, owing to a selective effect of octylglucoside at both solubilization and reconstitution steps. None of the various divalent cations used could dissipate the pH gradient of control liposomes of soybean lipids, unless the divalent/H+ exchanger A23187 was added, whereas a rapid dissipation of the pH gradient was observed with reconstituted proteoliposomes from lutoid proteins, with the cation selectivity sequence Zn2+ > Cd2+ > Mg2+ in the millimolar concentration range. The divalent ions Ca2+, Ba2+, and Mn2+ were incapable of generating a H+ efflux in reconstituted proteoliposomes, whereas both Mg2+/H+ and Ca2+/H+ exchanges were observed in lyophilized-resuspended lutoids. Therefore, the lutoid membrane seems to contain separate Mg2+/H+ and Ca2+/H transport systems, the latter being eliminated during the solubilization/reconstitution of lutoid membrane proteins.