scholarly journals The reversibility of active sulphate transport in membrane vesicles of Paracoccus denitrificans

1975 ◽  
Vol 150 (3) ◽  
pp. 527-536 ◽  
Author(s):  
J N Burnell ◽  
P John ◽  
F R Whatley
Keyword(s):  
Plasma Membrane ◽  
Active Transport ◽  
Paracoccus Denitrificans ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Thiol Group ◽  
Whole Cells ◽  
Sulphate Transport ◽  
Proton Symport ◽  
Inside Out

An uncoupler-sensitive active transport of sulphate into membrane vesicles prepared from the plasma membrane of Paracoccus denitrificans (previously Micrococcus denitrificans) can be driven by respiration or by a trans-membrane pH gradient (alkaline inside) generated by the addition either of KCL (in the presence of nigericin) or of NH4CL. Valinomycin does not substitute for nigericin. Respiration-driven transport is observed in right-side-out vesicles but not in inside-out vesicles, whereas transport driven by the addition of KCL (in the presence of nigericin) or of NH4CL is observed in both types of membrane vesicle. The active transport of sulphate into these vesicles is shown to be carrier-mediated by its sensitivity to thiol-group reagents. It is proposed that the sulphate carrier in the plasma membrane of P. denitrificans operates by a mechanism of electroneutral proton symport, and is capable of actively transporting sulphate in either direction across the plasma membrane, but that in whole cells respiration-driven proton expulsion drives the accumulative uptake of sulphate.

scholarly journals ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

1969 ◽  
Vol 41 (2) ◽  
pp. 378-392 ◽  
Author(s):  
Charles W. Boone ◽  
Lincoln E. Ford ◽  
Howard E. Bond ◽  
Donald C. Stuart ◽  
Dianne Lorenz
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicle ◽  
Whole Cells ◽  
Continuous Sucrose Gradient

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with 125I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.

scholarly journals Sodium-gradient-stimulated transport of l-alanine by plasma-membrane vesicles isolated from liver parenchymal cells of fed and starved rats. Crucial role of the adrenal glucocorticoids

1982 ◽  
Vol 208 (3) ◽  
pp. 685-693 ◽  
Author(s):  
Dennis C. Quinlan ◽  
C. Gordon Todderud ◽  
Darshan S. Kelley ◽  
Rolf F. Kletzien
Keyword(s):  
Plasma Membrane ◽  
Active Transport ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicle ◽  
Plasma Membrane Vesicles ◽  
Free System ◽  
Liver Parenchymal ◽  
Alanine Transport ◽  
Parenchymal Cells

The ability of liver efficiently to take up amino acids, particularly l-alanine, during starvation was studied in a cell-free system by isolating plasma-membrane vesicles in a transport-competent state from rat liver parenchymal cells. These membrane vesicles have the capacity to accumulate l-alanine against an apparent concentration gradient when exposed to an artificial and transient transmembrane Na+ gradient (extravesicular Na+ concentration greater than inside). The rate of accumulation of l-alanine is dependent on the plasma-membrane vesicle concentration, and the steady-state concentration attained is inversely related to the osmolarity of the medium. The Na+-mediated stimulation is not exhibited if the membrane vesicles are pre-equilibrated with NaCl, if K+ or Li+ are substituted for Na+, or if SO42− replaces Cl− as the counterion. The apparent active transport of l-alanine into the membrane vesicles appears to occur by an electrogenic mechanism: (1) the use of NaSCN significantly heightens the early concentrative phase of transport when compared with the effect of NaCl; (2) an enhanced active transport is also observed when a valinomycin-induced K+ efflux occurs concomitant with Na+ and l-alanine influx. Plasma-membrane vesicles isolated from liver parenchymal cells of a 24 h-starved rat exhibit an initial l-alanine transport rate that is 3–4 times that for membrane vesicles derived from a fed animal. The increased rate of l-alanine transport by plasma-membrane vesicles from starved animals can be obliterated by adrenalectomy and restored by administration of glucocorticoid. These results establish that stimulation of the gluconeogenic pathway by starvation involves a plasma-membrane-localized change affecting l-alanine transport which is regulated in part by the glucocorticoid hormones.

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.

The iodide channel of the thyroid: a plasma membrane vesicle study

1992 ◽  
Vol 263 (3) ◽  
pp. C590-C597 ◽  
Author(s):  
P. Golstein ◽  
M. Abramow ◽  
J. E. Dumont ◽  
R. Beauwens
Keyword(s):  
Plasma Membrane ◽  
Chloride Transport ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Anion Channel ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Vesicle ◽  
Plasma Membrane Vesicles ◽  
Bovine Thyroid ◽  
Set Up

The uptake of radioactive iodide or chloride by plasma membrane vesicles of bovine thyroid was studied by a rapid filtration technique. A Na(+)-I- cotransport was demonstrated. When this Na(+)-I- cotransport is inactive (i.e., at 4 degrees C and in the absence of Na+), an uptake of iodide above chemical equilibrium could be induced, driven by the membrane potential. The latter was set up by allowing potassium to diffuse into the membrane vesicles in the presence of valinomycin and of an inward K+ gradient. This potential difference (positive inside) induced the uptake of iodide (or other anion present). The data support the existence of two anionic channels. The first one, observed at low near-physiological iodide concentration (micromolar range), which exhibits a high permeability and specificity for iodide (hence called the iodide channel), has a Km of 70 microM. The other one appears similar to the epithelial anion channel as described by Landry et al. (J. Gen. Physiol. 90: 779-798, 1987); it is still about fourfold more permeable to iodide than to chloride and presents a Km of 33 mM. Under physiological conditions the latter channel would mediate chloride transport, and the iodide channel, which is proposed to be restricted to the apical plasma membrane domain of the thyrocyte, transports iodide from the cytosol to the colloid space.

scholarly journals Phospholipase D2 Localizes to the Plasma Membrane and Regulates Angiotensin II Receptor Endocytosis

2004 ◽  
Vol 15 (3) ◽  
pp. 1024-1030 ◽  
Author(s):  
Guangwei Du ◽  
Ping Huang ◽  
Bruce T. Liang ◽  
Michael A. Frohman
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Dominant Negative ◽  
Secretory Cells ◽  
Resting Cells ◽  
Angiotensin Ii Receptor ◽  
Multiple Cell

Phospholipase D (PLD) is a key facilitator of multiple types of membrane vesicle trafficking events. Two PLD isoforms, PLD1 and PLD2, exist in mammals. Initial studies based on overexpression studies suggested that in resting cells, human PLD1 localized primarily to the Golgi and perinuclear vesicles in multiple cell types. In contrast, overexpressed mouse PLD2 was observed to localize primarily to the plasma membrane, although internalization on membrane vesicles was observed subsequent to serum stimulation. A recent report has suggested that the assignment of PLD2 to the plasma membrane is in error, because the endogenous isoform in rat secretory cells was imaged and found to be present primarily in the Golgi apparatus. We have reexamined this issue by using a monoclonal antibody specific for mouse PLD2, and find, as reported initially using overexpression studies, that endogenous mouse PLD2 is detected most readily at the plasma membrane in multiple cell types. In addition, we report that mouse, rat, and human PLD2 when overexpressed all similarly localize to the plasma membrane in cell lines from all three species. Finally, studies conducted using overexpression of wild-type active or dominant-negative isoforms of PLD2 and RNA interference-mediated targeting of PLD2 suggest that PLD2 functions at the plasma membrane to facilitate endocytosis of the angiotensin II type 1 receptor.

scholarly journals Analysis of the Antimalarial Drug Resistance Protein Pfcrt Expressed in Yeast

2002 ◽  
Vol 277 (51) ◽  
pp. 49767-49775 ◽  
Author(s):  
Hanbang Zhang ◽  
Ellen M. Howard ◽  
Paul D. Roepe
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Chloroquine Resistance ◽  
Malarial Parasite ◽  
Plasma Membrane Vesicle ◽  
Wild Type ◽  
Stem Loop

Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) inPlasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000)Mol. Cell6, 861–871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane ofSaccharomyces cerevisiaeandPichia pastorisyeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild typeversusCQR-conferring mutant Pfcrt function, which may include effects on H+transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000)Mol. Biochem. Parasitol.110, 107–124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of importantP. falciparumtransport proteins and membrane associated vaccine candidates.

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