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

1997 ◽  
Vol 321 (2) ◽  
pp. 487-495 ◽  
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.

scholarly journals Maltose/proton co-transport in Saccharomyces cerevisiae. Comparative study with cells and plasma membrane vesicles

1992 ◽  
Vol 284 (2) ◽  
pp. 441-445 ◽  
Author(s):  
C C M Van Leeuwen ◽  
R A Weusthuis ◽  
E Postma ◽  
P J A Van den Broek ◽  
J P Van Dijken
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Maltose Transport

Maltose/proton co-transport was studied in intact cells and in plasma membrane vesicles of the yeast Saccharomyces cerevisiae. In order to determine uphill transport in vesicles, plasma membranes were fused with proteoliposomes containing cytochrome c oxidase as a proton-motive force-generating system. Maltose accumulation, dependent on the electrical and pH gradients, was observed. The initial uptake velocity and accumulation ratio in vesicles proved to be dependent on the external pH. Moreover, kinetic analysis of maltose transport showed that Vmax. values greatly decreased with increasing pH, whereas the Km remained virtually constant. These observations were in good agreement with results obtained with intact cells, and suggest that proton binding to the carrier proceeds with an apparent pK of 5.7. The observation with intact cells that maltose is co-transported with protons in a one-to-one stoichiometry was ascertained in the vesicle system by measuring the balance between proton-motive force and the chemical maltose gradient. These results show that maltose transport in vesicles prepared by fusion of plasma membranes with cytochrome c oxidase proteoliposomes behaves in a similar way as in intact cells. It is therefore concluded that this vesicle model system offers a wide range of new possibilities for the study of maltose/proton co-transport in more detail.

Pore-Spanning Plasma Membranes Derived from Giant Plasma Membrane Vesicles

2021 ◽  
Author(s):  
Nikolas K. Teiwes ◽  
Ingo Mey ◽  
Phila C. Baumann ◽  
Lena Strieker ◽  
Ulla Unkelbach ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles

scholarly journals Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

1996 ◽  
Vol 314 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R. Alexander BLACKWOOD ◽  
James E. SMOLEN ◽  
Ronald J. HESSLER ◽  
Donna M. HARSH ◽  
Amy TRANSUE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Phospholipid Vesicle ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Fluorescent Marker ◽  
Whole Cells ◽  
Specific Granules ◽  
Neutrophil Degranulation

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.

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

1998 ◽  
Vol 275 (4) ◽  
pp. C995-C1008 ◽  
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.

Two Phases Of Ferricyanide Reductase Activity In Ehrlich Cell Plasma Membranes

1992 ◽  
Vol 47 (11-12) ◽  
pp. 929-931 ◽  
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

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

1997 ◽  
Vol 273 (4) ◽  
pp. G842-G848 ◽  
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.

scholarly journals 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

1991 ◽  
Vol 273 (2) ◽  
pp. 429-434 ◽  
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.

scholarly journals K+/H+ antiport in the tobacco hornworm midgut: the K(+)-transporting component of the K+ pump.

1994 ◽  
Vol 196 (1) ◽  
pp. 361-373 ◽  
Author(s):  
A Lepier ◽  
M Azuma ◽  
W R Harvey ◽  
H Wieczorek
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Tobacco Hornworm ◽  
Proton Motive Force ◽  
Motive Force ◽  
Apical Plasma Membrane ◽  
Lectin Staining ◽  
Sds Page ◽  
K Transport ◽  
Midgut Lumen

The midgut of the tobacco hornworm secretes K+ across the apical plasma membrane of its goblet cells. This secondary K+ transport results from K+/H+ antiport energized by the proton-motive force generated by a primary, H(+)-transporting plasma membrane V-ATPase. Thus, the lepidopteran midgut constitutes a well-established example of the emerging concept that the proton-motive force is an alternative to the classical sodium-motive force for the energization of animal plasma membranes. K+/H+ antiport in the tobacco hornworm midgut is electrophoretic, exchanging 2H+ for 1K+. Under physiological conditions, it is energized by the voltage component of the proton-motive force. The strong coupling of electrophoretic K+/2H+ antiport with the electrogenic V-ATPase provides, in principle, the minimal device for the alkalization of the midgut lumen to pH values higher than 11. K+/H+ antiport is insensitive to bafilomycin A1, but is inhibited by amiloride or Concanavalin A. Lectin staining of blots after SDS-PAGE revealed several glycosylated polypeptides in the goblet cell apical membrane which are not part of the V-ATPase and thus are candidates for the antiporter protein. Current efforts are focused on the isolation of the K+/H+ antiporter.

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