Preparation Of Inside-out Vesicles Research Articles

Calcium transport in gill plasma membranes of the crab Carcinus Maenas: Evidence for carriers driven by ATP and A Na+ Gradient

A procedure was developed for the preparation of inside-out vesicles from plasma membranes isolated from the branchial epithelium of the green shore crab Carcinus maenas (L.). Procedures normally applied to fish branchial epithelium required the introduction of an additional hypotonic shock to obtain a preparation containing 22% inside-out vesicles, 33% right-side-out vesicles and 45% leaky membrane fragments. In such membrane preparations, the first direct evidence for uphill (against a [Ca2+] gradient) ATP-dependent and Na(+)-gradient-dependent Ca2+ transport in crustacean gills was found. The affinity for Ca2+ of the ATP-driven Ca2+ transporter was 149 nmol l-1 and that of the Na+/Ca2+ exchanger was 1.78 mumol l-1; the Vmax values were 1.73 and 9.88 nmol min-1 mg-1 protein respectively. The relative importance of these carriers for Ca2+ transport in the branchial epithelium of the crab is evaluated on the basis of their calcium kinetics.

Electron microscopy of native and artificial methylreductase high-molecular-weight complexes in strain Gö 1 and Methanococcus voltae

The preparation of inside-out vesicles from methanogenic bacteria with protein cell walls was improved with regard to the preservation of structure and localization of membrane-bound proteins. Complexes similar to the methanoreductosome in the methanogenic bacterium Gö 1 [1] were also found attached to the inner aspect of the cytoplasmic membrane of Methanococcus voltae. Methanoreductosomes were purified from crude extracts of Gö 1-cells by affinity chromatography. Under specific conditions at high protein concentrations methyl-CoM-methylreductase molecules isolated from Gö 1-cells could be reassociated to spherical complexes of various sizes, with an appearance similar to that of methanoreductosomes isolated from strain Gö 1.

ATP synthesis coupled to methane formation from methyl‐CoM and H2 catalyzed by vesicles of the methanogenic bacterial strain Gö1

Methanogenesis from methyl-CoM and H2, as catalyzed by inside-out vesicle preparations of the methanogenenic bacterium strain Gö1, was associated with ATP synthesis. That this ATP synthesis proceeded via an uncoupler-sensitive transmembrane proton gradient was concluded from the following results: 1. Various inhibitors that affected methane formation (e.g. 2-bromomethanesulfonate) also prevented ATP synthesis. 2. The protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile, in combination with the K+ ionophore valinomycin, inhibited ATP synthesis completely without affecting methanogenesis. 3. The ATP synthase inhibitor diethylstilbestrol inhibited ATP synthesis. 4. Addition of the detergent sulfobetaine inhibited both methane formation and ATP synthesis; the former but not the latter could be restored by adding titanium(III) citrate as electron donor. In addition it was shown that ATP synthesis could also be driven by transmembrane proton gradients artificially imposed on the vesicles. Furthermore net methanogenesis-dependent ATP formation was shown by measuring [32P]phosphate incorporation.

An estimate of the number of Ca 2+-dependent K + channels in the human red cell

An original approach has been designed to count Ca 2+-dependent K + channels in the human red cell using a preparation of inside-out vesicles. The relative frequency of vesicles having no K + channels is estimated from the fraction of 42K + (or 86Rb +) which is not released from loaded vesicles on maximal stimulation with Ca 2+. The mean number of channels per vesicle is then calculated from this figure assuming a Poisson distribution for the K + channels. From this value and the mean vesicular radius, computed from the volume / surface ratio, the mean number of channels per cell can be estimated. A value of 142 ± 27 ( mean ± S.E. ) was obtained, which is well above that estimated by comparison of unitary conductance and tracer equilibration rate measurements (about 10 channels / cell, Grygorczyk, R. Schwarz, W. and Passow, H. (1984) Biophys. J. 45, 693–698), but compares favourably with the channel density inferred from comparison with the number of Na + pumps in a similar preparation of inside-out vesicles (100–200 / cell, Lew, V.L., Muallem, S. and Seymour, C.A. (1982) Nature 296, 742–744). The procedure described here can be considered for general application as an alternative to other known procedures.

Association of spectrin with desmin intermediate filaments

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament-erythrocyte inside-out vesicle preparations. Desmin filaments bound to erythrocyte inside-out vesicles in a spectrin-dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration-dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.

Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid

The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins. This pathway has been characterized further by studying the activation and incorporation of [9,10(n)- 3H]oleic acid and transesterification of [1- 14C]oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts. Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars. Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid. Compared to ghosts, the ability of inside-out vesicles to activate and incorporate [9,10(n)- 3H]oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify [1- 14C]oleoyl-CoA to phospholipid is diminished by over 50%. These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities. This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation.

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)

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.

Lectins as biochemical agents for the isolation of sealed membrabe vesicles of defined polarity

The asymmetric distribution of carbohydrate on biological membranes has provided the basis for the development of lectin-affinity methodology which permits the isolation of sealed, inside-out membrane fractions from heterogeneous populations of vesicles. Optimal conditions for these separations have been assessed employing purified right-side-out and inside-out vesicles derived from the plasma membrane of human erythrocytes as a model system. In this special case, homogeneous populations of defined polarity can be produced by varying the ionic conditions during formation of the vesicles. Surface-specific enzymic markers exist also for monitoring the integrity and orientation of a given population. Multivalent lectins such as wheat germ agglutinin and soya bean agglutinin, which induce direct agglutination of erythrocyte membrane fragments containing accessible carbohydrate residues, selectively remove more than 90% of right-side-out and non-sealed membrane from mixed population, a reaction which is inhibited by GluNAc or GalNAc, respectively. Non-agglutinating lectins, e.g. concanavalin A, immobilized on an inert matrix such as Sepharose 4B, may be employed to adsorb out specifically vesicles with exposed glycopeptides on their surface. In this technique, it is necessary normally to remove the non-sealed membranes on Dextran density gradients prior to the final preparation of inside-out vesicles on Con A-Sepharose. Finally, selective immunoprecipitation of fragments with accessible sugars may also be achieved after treatment with a non-agglutinating lectin (concanavalin A) followed by incubation with anti-concanavalin A IgG which promotes rapid aggregation of membrane containing exposed receptors for the lectin. These procedures should prove generally suitable for the isolation of tightly-sealed, inside-out membrane populations in a variety of biological systems. Pure populations of vesicles, exhibiting reversed polarity, are valuable in surface-labelling studies for investigating the structure, function and transmembrane distribution of integral membrane proteins/glycoproteins.

Simple and rapid purification of inside-out vesicles from human erythrocytes

The preparation of inside-out vesicles from human erythrocytes requires their separation from contaminating right-side-out vesicles. We have taken advantage of the fact that there are no glycoproteins on the internal side of the erythrocyte membrane; therefore, inside-out vesicles do not interact with the lectin, concanavalin A, while right-side-out vesicles do interact. A concanavalin A-cellulose affinity matrix has been utilized to separate easily inside-out vesicles with a purity comparable to those prepared by prolonged centrifugation.

A mechanism for the formation of inside-out membrane vesicles. Preparation of inside-out vesicles from membrane-paired randomized chloroplast lamellae

Inside-out thylakoid membrane vesicles can be isolated by aqueous polymer two-phase partition of Yeda press-fragmented spinach chloroplasts (Andersson, B. and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472). The mechanism for their formation has been investigated by studying the yield of inside-out vesicles after various treatments of the chloroplasts prior to fragmentation. No inside-out vesicles were isolated during phase partitioning if the chloroplasts had been destacked in a low-salt medium prior to the fragmentation. Only in those cases where the chloroplast lamellae had been stacked by cations or membrane-paired by acidic treatment did we get any yield of inside-out vesicles. Thus, the intrinsic properties of chloroplast thylakoids seem to be such that they seal into right-side out vesicles after disruption unless they are in an appressed state. This favours the following mechanism for the formation of inside-out thylakoids. After press treatment, a ruptured membrane still remains appressed with an adjacent membrane. Resealing of such an appressed membrane pair would result in an inside-out vesicle. If the compartmentation of chloroplast lamellae into appressed grana and unappressed stroma lamellae is preserved by cations before fragmentation, the inside-out vesicles are highly enriched in photosystem II. This indicates a granal origin which is consistent with the proposed model outlined. Inside-out vesicles possessing photosystem I and II properties in approximately equal proportions could be obtained by acid-induced membrane-pairing of chloroplasts which had been destacked and randomized prior to fragmentation. Since this new preparation of inside-out thylakoid vesicles also exposes components derived from the stroma lamellae it complements the previous preparation. It is suggested that fragmentation of paired membranes followed by phase partitioning should be a general method of obtaining inside-out vesicles from membranes of various biological sources.

Preparation of inside-out vesicles of pig lymphocyte plasma membrane.

Between 30 and 50% of pig lymphocyte plasma membrane vesicles were not bound by concanavalin A (Con A)-Sepharose. Various results suggest that the Con A-unretarded fraction represents "inside-out" membrane vesicles. First, an alternative cell surface ligand, anti-lymphocytic serum, gave a similar fractionation to Con A. Second, lack of binding by Con A was not due to lack of carbohydrate or to masking of carbohydrate by extraneous protein, because the unfractionated membrane and the unretarded fraction had similar carbohydrate and polypeptide compositions. Third although the carbohydrate of the unretarded membrane vesicles was accessible to 125I-labelled Con A and to release by soluble trypsin, it was not accessible to ferritin-Con A or trypsin-Sepharose. Fourth, antisera against the external surface of the Con A-unretarded vesicles strongly agglutinated the unretarded membrane, but caused negligible agglutination of whole lymphocytes. When attached to Sepharose these antisera bound all of the Con A-unretarded fraction, but failed to bind the membrane that adhered to Con A-Sepharose.

Localization of enzymes involve in polyphosphoinositide metabolism on the cytoplasmic surface of the human erythrocyte membrane

1. 1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. 2. Incubation of vesicles with [γ- 32P]ATP at 37 °C for periods of up to 1 h did not change the topography or the permeability of the vesicles. 3. 3. Vesicles incorporated labeled phosphate from [γ- 32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side-out vesicles were not as active. 5. 5. Impermeable right-side-out vesicles, treated with 0.01% saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane.