Pore-Spanning Plasma Membranes Derived from Giant Plasma Membrane Vesicles

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

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Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.4.c995 ◽

1998 ◽

Vol 275 (4) ◽

pp. C995-C1008 ◽

Cited By ~ 49

Author(s):

Christie Cefaratti ◽

Andrea Romani ◽

Antonio Scarpa

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Intracellular Signaling ◽

Mammalian Cells ◽

Plasma Membranes ◽

Membrane Vesicles ◽

Channel Blockers ◽

Transport Mechanisms ◽

Plasma Membrane Vesicles ◽

New Information

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

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Two Phases Of Ferricyanide Reductase Activity In Ehrlich Cell Plasma Membranes

Zeitschrift für Naturforschung C ◽

10.1515/znc-1992-11-1223 ◽

1992 ◽

Vol 47 (11-12) ◽

pp. 929-931 ◽

Cited By ~ 5

Author(s):

Antonio del Castillo-Olivares ◽

Javier Márquez ◽

Ignacio Núñez de Castro ◽

Miguel Angel Medina

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Reductase Activity ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Cell Plasma Membrane ◽

Ferricyanide Reductase ◽

Ehrlich Cell ◽

Cell Plasma ◽

Two Phases

Ehrlich cell plasma membrane vesicles have a ferricyanide reductase activity that shows two phases. These two phases were kinetically characterized. Evidence is presented for a differential effect of trypsin on both phases

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cAMP increases liver Na+-taurocholate cotransport by translocating transporter to plasma membranes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1997.273.4.g842 ◽

1997 ◽

Vol 273 (4) ◽

pp. G842-G848 ◽

Cited By ~ 14

Author(s):

Sunil Mukhopadhayay ◽

M. Ananthanarayanan ◽

Bruno Stieger ◽

Peter J. Meier ◽

Frederick J. Suchy ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Fusion Protein ◽

Protein Kinase A ◽

Plasma Membranes ◽

Membrane Vesicles ◽

Rat Hepatocytes ◽

Plasma Membrane Vesicles ◽

Short Term ◽

Kinase A

Adenosine 3′,5′-cyclic monophosphate (cAMP), acting via protein kinase A, increases transport maximum of Na+-taurocholate cotransport within 15 min in hepatocytes (S. Grüne, L. R. Engelking, and M. S. Anwer. J. Biol. Chem. 268: 17734–17741, 1993); the mechanism of this short-term stimulation was investigated. Cycloheximide inhibited neither basal nor cAMP-induced increases in taurocholate uptake in rat hepatocytes, indicating that cAMP does not stimulate transporter synthesis. Studies in plasma membrane vesicles showed that taurocholate uptake was not stimulated by the catalytic subunit of protein kinase A but was higher when hepatocytes were pretreated with cAMP. Immunoblot studies with anti-fusion protein antibodies to the cloned Na+-taurocholate cotransport polypeptide (Ntcp) showed that pretreatment of hepatocytes with cAMP increased Ntcp content in plasma membranes but not in homogenates. Ntcp was detected in microsomes, endosomes, and Golgi fractions, and cAMP pretreatment resulted in a decrease only in endosomal Ntcp content. It is proposed that cAMP increases transport maximum of Na+-taurocholate cotransport, at least in part, by translocating Ntcp from endosomes to plasma membranes.

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Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis

Biochemical Journal ◽

10.1042/bj3210487 ◽

1997 ◽

Vol 321 (2) ◽

pp. 487-495 ◽

Cited By ~ 12

Author(s):

Peter J. A. van den BROEK ◽

Angeline E. van GOMPEL ◽

Marijke A. H. LUTTIK ◽

Jack T. PRONK ◽

Carla C. M. van LEEUWEN

Keyword(s):

Plasma Membrane ◽

Candida Utilis ◽

Plasma Membranes ◽

Membrane Vesicles ◽

Proton Motive Force ◽

Motive Force ◽

Transport Systems ◽

Sugar Accumulation ◽

Plasma Membrane Vesicles ◽

Maltose Transport

Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome coxidase as a proton-motive-force-generating system. Addition of reduced cytochrome cgenerated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40Ő50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+Őglucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+Őmaltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilisthe transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.

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The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib β-subunit

Biochemical Journal ◽

10.1042/bj2730429 ◽

1991 ◽

Vol 273 (2) ◽

pp. 429-434 ◽

Cited By ~ 10

Author(s):

A Darnanville ◽

R Bredoux ◽

K J Clemetson ◽

N Kieffer ◽

N Bourdeau ◽

...

Keyword(s):

Plasma Membrane ◽

Time Course ◽

Plasma Membranes ◽

Polyclonal Antibodies ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Glycoprotein Ib ◽

Dependent Protein ◽

Dose Dependent ◽

Fold Stimulation

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.

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Bicarbonate-chloride exchange in gill plasma membranes of blue crab

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1985.249.5.r544 ◽

1985 ◽

Vol 249 (5) ◽

pp. R544-R550 ◽

Cited By ~ 1

Author(s):

S. H. Lee ◽

J. B. Pritchard

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

External Medium ◽

Callinectes Sapidus ◽

Exchange Process ◽

Membrane Vesicles ◽

Disulfonic Acid ◽

Plasma Membrane Vesicles ◽

Low Salinity ◽

Chloride Exchange

The uptake of chloride was studied in gill plasma membrane vesicles from low-salinity-adapted blue crabs (Callinectes sapidus). Cl- uptake was not Na+ dependent. However, when a HCO-3 gradient (in greater than out) was imposed across the membrane, a transient overshoot of about 2.5-fold was produced. Approximately 90% of the Cl- uptake reflected entry into the osmotically active intravesicular space. Cl- itself, nitrate, hydroxyl, and sulfite could substitute for HCO-3. The HCO-3/Cl- exchange process appeared to saturate at higher concentrations of either HCO-3 or Cl-. The apparent Km for Cl- was 15 mM. HCO-3-dependent Cl- uptake was significantly inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and thiocyanate but not by amiloride, furosemide, or ouabain. Alterations in membrane potential had no effect on Cl- uptake. Addition of Cl- or HCO-3 to the external medium also accelerated efflux of 36Cl- and H14CO-3 from preloaded vesicles. These results indicate that the uptake of Cl- by the crab gill plasma membrane is a carrier-mediated Na+-independent anion exchange process.

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myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes

Biochemical Journal ◽

10.1042/bj2360037 ◽

1986 ◽

Vol 236 (1) ◽

pp. 37-44 ◽

Cited By ~ 42

Author(s):

D M Delfert ◽

S Hill ◽

H A Pershadsingh ◽

W R Sherman ◽

J M McDonald

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Plasma Membranes ◽

Initial Rate ◽

Membrane Vesicles ◽

Heavy Fraction ◽

Differential Centrifugation ◽

Plasma Membrane Vesicles ◽

Inositol 1,4,5 Trisphosphate ◽

Uptake And Release

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.

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Isolation and characterization of plasma membranes from bovine carotid arteries

AJP Cell Physiology ◽

10.1152/ajpcell.1986.250.1.c65 ◽

1986 ◽

Vol 250 (1) ◽

pp. C65-C75 ◽

Cited By ~ 9

Author(s):

R. V. Sharma ◽

R. C. Bhalla

Keyword(s):

Plasma Membrane ◽

Cytochrome C ◽

Membrane Fraction ◽

Plasma Membranes ◽

Carotid Arteries ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Cytochrome C Reductase ◽

Plasma Membrane Fraction

A plasma membrane fraction from bovine carotid arteries has been isolated by extraction of a crude microsomal fraction with a low-ionic-strength buffer containing ATP and Ca2+. This step was followed by sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membrane vesicles were enriched 60- to 80-fold in Na+-K+-adenosinetriphosphatase, 5'-nucleotidase, and phosphodiesterase I activities. The final yields of these marker enzymes were 12-18% of the total activities in the postnuclear supernatant, and the protein yield was 100-120 micrograms/g wet wt of carotid arteries. Contamination of the plasma membrane fraction by mitochondria and sarcoplasmic reticulum was low as judged by low activities of succinate--cytochrome-c reductase and NADPH--cytochrome-c reductase, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with smooth muscle-specific actin antibodies showed that the plasma membrane fraction was substantially free from myosin and actin contamination. The plasma membrane vesicles accumulated Ca2+ in the presence of ATP, and the accumulation was increased by calmodulin. Ca2+ accumulated in the presence or absence of calmodulin could be released almost completely from the vesicles by the addition of the Ca2+ ionophore A23187 but not by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca2+ uptake in the presence of ATP is intravesicular. The effects of phosphate and oxalate on Ca2+ uptake in the plasma membranes were different from one another. Phosphate increased Ca2+ uptake in a concentration- and time-dependent manner, and the increase in Ca2+ uptake could be observed as early as 1 min. On the other hand, oxalate at concentrations up to 5 mM did not increase Ca2+ uptake significantly during the 30-min incubation. These plasma membranes can prove useful for the study of ion transport across plasma membranes, hormone binding, characterization of calcium channels, and preparation of antibodies against plasma membrane proteins.

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Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

The Journal of Cell Biology ◽

10.1083/jcb.86.1.21 ◽

1980 ◽

Vol 86 (1) ◽

pp. 21-28 ◽

Cited By ~ 82

Author(s):

M S Klempner ◽

R B Mikkelsen ◽

D H Corfman ◽

J André-Schwartz

Keyword(s):

Plasma Membrane ◽

Human Neutrophil ◽

Plasma Membranes ◽

Hydrolytic Enzymes ◽

Membrane Vesicles ◽

Galactose Oxidase ◽

High Yield ◽

Differential Centrifugation ◽

Plasma Membrane Vesicles ◽

Equilibrium Ultracentrifugation

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

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