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

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.

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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

Biochemical Journal ◽

10.1042/bj2080685 ◽

1982 ◽

Vol 208 (3) ◽

pp. 685-693 ◽

Cited By ~ 12

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.

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Analysis of the Antimalarial Drug Resistance Protein Pfcrt Expressed in Yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m204005200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49767-49775 ◽

Cited By ~ 54

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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ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

The Journal of Cell Biology ◽

10.1083/jcb.41.2.378 ◽

1969 ◽

Vol 41 (2) ◽

pp. 378-392 ◽

Cited By ~ 52

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.

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In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽

10.1182/blood.v67.5.1454.1454 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1454-1459 ◽

Cited By ~ 6

Author(s):

L Feldman ◽

CM Cohen ◽

N Dainiak

Keyword(s):

Plasma Membrane ◽

Membrane Vesicle ◽

In Vitro Release ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Wide Range ◽

Dose Dependent Fashion ◽

Membrane Bound ◽

Opposing Effects

Abstract In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

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Smooth muscle membrane vesicle orientation: a study on intactness and sidedness of rat myometrium plasma membrane vesicles

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y80-183 ◽

1980 ◽

Vol 58 (10) ◽

pp. 1202-1211 ◽

Cited By ~ 18

Author(s):

A. K. Grover ◽

J. Crankshaw ◽

R. E. Garfield ◽

E. E. Daniel

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Shock Treatment ◽

Galactose Oxidase ◽

Muscle Membrane ◽

Plasma Membrane Vesicles ◽

Binding Studies ◽

Hypotonic Shock

Plasma membrane vesicles of rat myometrium were prepared in media containing 240 mM sucrose. The vesicles were exposed to isotonic, hypertonic, and hypotonic sucrose concentrations, fixed, sectioned, and studied using the electron microscope. The vesicles fixed in isotonic media were circular in appearance. Vesicles fixed in hypertonic media were distorted and showed a reduced volume to surface ratio consistent with the hypothesis that >80% of the vesicles were osmotically active to sucrose. Cationized ferritin binding studies and Ca binding and release studies were also consistent with this finding. Exposure to hypotonic media also yielded membranes with distorted profiles indicating that they had been ruptured. [3H]Sucrose trapping experiments revealed that the vesicles had an internal volume of 1.20–1.44 mL/g protein. Hypotonic shock treatment reduced this intravesicular volume to 0.20–0.28 mL/g protein. The hypotonic shock treatment also led to enhanced galactose oxidase catalyzed Na3B3H4 labelling of the membranes and to increased K+-activated ouabain-sensitive p-nitrophenyl phosphatase activity. The enhancement was the same (55 ± 10%) in the various membrane preparations for both the parameters. The data are interpreted to conclude that the rat myometrium plasma membrane vesicles consisted of 20% broken vesicles and equal proportions of intact vesicles of inside-out and rightside-out orientations.

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Apical plasma membrane vesicles formed from organ donor colon demonstrate Na+ and H+ conductances and Na+H+ exchange

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(90)92042-x ◽

1990 ◽

Vol 167 (2) ◽

pp. 438-443 ◽

Cited By ~ 20

Author(s):

James M. Harig ◽

Pradeep K. Dudeja ◽

Strawn M. Knaup ◽

Jihad Shoshara ◽

Krishnamurthy Ramaswamy ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Vesicles ◽

Organ Donor ◽

Apical Plasma Membrane ◽

Plasma Membrane Vesicles

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In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽

10.1182/blood.v67.5.1454.bloodjournal6751454 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1454-1459

Author(s):

L Feldman ◽

CM Cohen ◽

N Dainiak

Keyword(s):

Plasma Membrane ◽

Membrane Vesicle ◽

In Vitro Release ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Wide Range ◽

Dose Dependent Fashion ◽

Membrane Bound ◽

Opposing Effects

In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

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