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.     

Sidedness of ceramide-phosphoethanolamine synthesis on rat liver plasma membrane

Phosphatidylethanolamine:ceramide-ethanolaminephosphotransferase catalyzes the synthesis of ceramide-ethanolamine, a sphingomyelin analogue. Its transverse localization in rat liver plasma membrane was studied by treating intact and deoxycholate- or Triton X-100-disrupted membrane vesicles with trypsin or bacterial protease. The latency of ATPase was preserved during protease treatment; its value was 80% in the membrane vesicles obtained by sucrose gradient procedure alone and 91.2% in the vesicles isolated after sucrose gradient plus two-phase partitioning. This suggested that membrane integrity was not altered and that 90% of the vesicles were right-side out. When the sucrose gradient was followed by the two-phase procedure, 62% of phosphatidylethanolamine:ceramide-ethanolamine-phosphotransferase was accessible to the protease action, but only 45% in vesicles obtained by sucrose gradient alone. Our results suggest that at least a sizable portion of the active center of the enzyme responsible of biosynthesis of ceramide-phosphoethanolamine is located on the external side of liver plasma membrane and that the other is embedded in the membrane interior and is not accessible to trypsin, even in the presence of detergent.     

Calcium uptake by placental plasma membrane vesicles

The Placental plasma membrane vesicles are capable of accumulating up to 190 mM Ca²⁺. This is 24-fold higher than the external Ca²⁺ concentration.This process is dependent on ATP hydrolysis by the placental Ca²⁺-ATPase.The $\text{P}{}_{\mathrm{i}}\text{Ca}$ ratio is dependent on the external Ca²⁺ concentration, and reaches the value of 2 at 10 mM Ca²⁺.Phosphate (5 mM) can double Ca²⁺ uptake when measured in the presence of 5 mM Ca²⁺.Mg²⁺; increased Ca²⁺ uptake only at low Ca²⁺ concentrations, and had no significant effect at 5 mM Ca²⁺.     

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.     

Calcium uptake by placental plasma membrane vesicles.

The placental plasma membrane vesicles are capable of accumulating up to 190 mM Ca2+. This is 24-fold higher than the external Ca2+ concentration. This process is dependent on ATP hydrolysis by the placental Ca2+-ATPase. The Pi/Ca ratio is dependent on the external Ca2+ concentration, and reaches the value of 2 at 10 mM Ca2+. Phosphate (5 mM) can double Ca2+ uptake when measured in the presence of 5 mM Ca2+. Mg2+ increased Ca2+ uptake only at low Ca2+ concentrations, and had no significant effect at 5 mM Ca2+.     

Giant plasma membrane vesicles to study plasma membrane structure and dynamics

The plasma membrane (PM) is a highly heterogenous structure intertwined with the cortical actin cytoskeleton and extracellular matrix. This complex architecture makes it difficult to study the processes taking place at the PM. Model membrane systems that are simple mimics of the PM overcome this bottleneck and allow us to study the biophysical principles underlying the processes at the PM. Among them, cell-derived giant plasma membrane vesicles (GPMVs) are considered the most physiologically relevant system, retaining the compositional complexity of the PM to a large extent. GPMVs have become a key tool in membrane research in the last few years. In this review, I will provide a brief overview of this system, summarize recent applications and discuss the limitations.     

Localization of intracellular zinc under conditions of altered permeability control of the plasma membrane

Intracellular zinc was located as electron dense granules associated with the plasma membrane, endoplasmic reticular membranes, mitochondrial membranes, nuclear membranes and chromatin in Zajdela ascitic hepatoma and AK5 macrophage ascitic tumour cells. The quantity of intracellular zinc estimated by atomic emission spectrometer was different in the two cell lines. However, after loss of permeability control by the plasma membrane, involving glutaraldehyde and heat-shock treatments, the quantity of intracellular zinc was increased to almost the same extent in both cases.     

Active uptake of sucrose by plant plasma membrane vesicles: determination of some important physical and energetical parameters

Changes in general protease activity and the levels of the zein storage proteins were monitored during germination and early seedling growth of maize ( Zea mays L . inbred A636). General endosperm endoprotease activity, measured in vitro using azocasein as a substrate, increased continuously to day 5 and remained high thereafter. The increase was in parallel with the loss of zein protein as determined by immunoblot analysis, with a total loss of detectable zein by 10 days after inhibition of the seeds. A method was developed for the specific in vitro assay of zein degrading activity by monitoring the release of soluble radioactivity from the immobilized storage protein. The in vitro pH optimum for both general protease and zein degrading activities in homogenates prepared from isolated endosperm was 4.0. However, the curve for general protease activity was asymmetrical suggesting the presence of more than 1 protease. This was verified by activity staining of gelatin-containing polyacrylamide gels, which suggested the presence of 3 major and 3 minor protease activities. Both general protease and zein-specific activities were enhanced more than 2-fold when 2-mercaptoethanol (ME) was included in the assays, and both were inhibited by fhiol-protease directed compounds. Most of the general protease activity but none of the zein-specific protease activity bound to con A-Sepharose. Both the con A-binding and nonbinding fractions were analyzed using gelatin activity gels. The 3 minor activity bands were present in the fraction which was specifically eluted from the con A column by a-methyl-mannoside, while the 3 major activity bands, corresponding to the zein-specific protease activity, did not bind to the immobilized lectin.     

Sealed inside-out and right-side-out plasma membrane vesicles : optimal conditions for formation and separation

Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of inside-out vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H⁺ pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% right-side-out vesicles, respectively, were obtained. ATPase activity and H⁺ pumping were both completely inhibited by vanadate (K_i ≈ 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore, the polypeptide patterns of the two fractions were close to identical, which shows that the vesicles differed in sidedness only. Thus, preparations of both inside-out and right-side-out plasma membrane vesicles are now available. This permits studies on transport, signal transduction mechanisms, enzyme topology, etc., using plasma membrane vesicles of either orientation.     

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

L-glutamate transport in renal plasma membrane vesicles

Summary This review describes the uptake of L-glutamate by well-characterized preparations of renal brush border (luminal) and baso-lateral membrane vesicles derived from the plasma membrane of the polar proximal tubular cell. L-glutamate is taken up against its concentration gradient, from both sides, by co-transport systems in which the movement of the amino acid into the cell is coupled to the influx of Na⁺ and efflux of K⁺ down their respective electrochemical gradients. The presence of these ion gradient-energized systems, specific for L-glutamate, may account for the exceedingly high intracellular concentration of this metabolically important amino acid in the renal tubule.     

Protective plant immune responses are elicited by bacterial outer membrane vesicles

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Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.