Basal-lateral and intracellular membrane populations of rat exorbital lacrimal gland

1983 ◽  
Vol 245 (1) ◽  
pp. G133-G142
Author(s):  
A. K. Mircheff ◽  
C. N. Conteas ◽  
C. C. Lu ◽  
N. A. Santiago ◽  
G. M. Gray ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Lacrimal Gland ◽  
Membrane Vesicle ◽  
Sedimentation Coefficient ◽  
Plasma Membrane Vesicle ◽  
Intracellular Membrane ◽  
Lateral Membrane ◽  
Specific Activities ◽  
Analytical Fractionation ◽  
Distinct Membrane

With the goal of isolating and identifying plasma membrane vesicle populations from epithelial cells of the rat exorbital lacrimal gland, we have designed an analytical fractionation of homogenates of the gland parenchyma. This fractionation utilizes separation procedures based on three independent physical properties of subcellular particles: sedimentation coefficient, density, and density after interaction of membrane cholesterol with digitonin. A commonly accepted marker for basal-lateral membranes, Na-K-ATPase, is associated with at least two physically distinct membrane populations. One population can be identified as basal-lateral membrane fragments on the basis of its fractional and specific contents of Na-K-ATPase; it accounts for 50% of the total Na-K-ATPase activity, enriched 29-fold with respect to the initial homogenate. With these values we calculate that the sample of basal membranes has been purified 60-fold with respect to the initial homogenate. The remaining Na-K-ATPase activity appears to be associated, at three- to fivefold lower specific activities, with intracellular membrane populations. We speculate that these populations have been derived from the Golgi complex.

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.

Translation initiation factors and active sites of protein synthesis co-localize at the leading edge of migrating fibroblasts

2011 ◽  
Vol 438 (1) ◽  
pp. 217-227 ◽  
Author(s):  
Mark Willett ◽  
Michele Brocard ◽  
Alexandre Davide ◽  
Simon J. Morley
Keyword(s):  
Protein Synthesis ◽  
Cell Migration ◽  
Active Sites ◽  
Membrane Vesicle ◽  
Leading Edge ◽  
Membrane Microdomains ◽  
Plasma Membrane Vesicle ◽  
Translational Machinery ◽  
Initiation Factors ◽  
Scanning Confocal Microscopy

Cell migration is a highly controlled essential cellular process, often dysregulated in tumour cells, dynamically controlled by the architecture of the cell. Studies involving cellular fractionation and microarray profiling have previously identified functionally distinct mRNA populations specific to cellular organelles and architectural compartments. However, the interaction between the translational machinery itself and cellular structures is relatively unexplored. To help understand the role for the compartmentalization and localized protein synthesis in cell migration, we have used scanning confocal microscopy, immunofluorescence and a novel ribopuromycylation method to visualize translating ribosomes. In the present study we show that eIFs (eukaryotic initiation factors) localize to the leading edge of migrating MRC5 fibroblasts in a process dependent on TGN (trans-Golgi network) to plasma membrane vesicle transport. We show that eIF4E and eIF4GI are associated with the Golgi apparatus and membrane microdomains, and that a proportion of these proteins co-localize to sites of active translation at the leading edge of migrating cells.

scholarly journals Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes

1986 ◽  
Vol 233 (3) ◽  
pp. 661-668 ◽  
Author(s):  
N Hack ◽  
M Croset ◽  
N Crawford
Keyword(s):  
Atpase Activity ◽  
Blood Platelets ◽  
Present Report ◽  
Surface Membrane ◽  
Storage Site ◽  
Intracellular Membrane ◽  
Bivalent Cation ◽  
Platelet Surface ◽  
Intracellular Membranes ◽  
Dependent Transport

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.

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.

Role of different Escherichia coli hydrogenases in H+ efflux and F1Fo-ATPase activity during glycerol fermentation at different pH values

2011 ◽  
Vol 31 (3) ◽  
pp. 179-184 ◽  
Author(s):  
Syuzanna Blbulyan ◽  
Arev Avagyan ◽  
Anna Poladyan ◽  
Armen Trchounian
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Membrane Vesicle ◽  
Wild Type ◽  
Glucose Fermentation ◽  
Glycerol Fermentation ◽  
Whole Cells ◽  
Ph Values ◽  
F1fo Atpase

Escherichia coli is able to ferment glycerol and produce H2 by different Hyds (hydrogenases). Wild-type whole cells were shown to extrude H+ through the F1Fo-ATPase and by other means with a lower rate compared with that under glucose fermentation. At pH 7.5, H+ efflux was stimulated in fhlA mutant (with defective transcriptional activator of Hyd-3 or Hyd-4) and was lowered in hyaB or hybC mutants (with defective Hyd-1 or Hyd-2) and hyaB hybC double mutant; DCCD (dicyclohexylcarbodi-imide)-sensitive H+ efflux was observed. At pH 5.5, H+ efflux in wild-type was lower compared with that at pH 7.5; it was increased in fhlA mutant and absent in hyaB hybC mutant. Membrane vesicle ATPase activity was lower in wild-type glycerol-fermented cells at pH 7.5 compared with that in glucose-fermented cells; 100 mM K+ did not stimulate ATPase activity. The latter at pH 7.5, compared with that in wild–type, was lower in hyaB and less in hybC mutants, stimulated in the hyaB hybC mutant and suppressed in the fhlA mutant; DCCD inhibited ATPase activity. At pH 5.5, the ATPase activities of hyaB and hybC mutants had similar values and were higher compared with that in wild-type; ATPase activity was suppressed in hyaB hybC and fhlA mutants. The results indicate that during glycerol fermentation, H+ was expelled also via F1Fo. At pH 7.5 Hyd-1 and Hyd-2 but not FhlA or Hyd-4 might be related to F1Fo or have their own H+-translocating ability. At pH 5.5, both Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.

Amiloride-inhibitable Na+ conductive pathways in alveolar type II pneumocytes

1991 ◽  
Vol 260 (2) ◽  
pp. L90-L96 ◽  
Author(s):  
S. Matalon ◽  
R. J. Bridges ◽  
D. J. Benos
Keyword(s):  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Na Channel ◽  
Plasma Membrane Vesicle ◽  
Alveolar Type ◽  
Type Ii ◽  
Type Ii Pneumocytes ◽  
The Difference ◽  
22Na Uptake ◽  
Na Uptake

The purpose of these studies was to document the existence of electrogenic Na+ uptake by membrane vesicles of rabbit alveolar type II (ATII) cells and the extent to which this process was inhibited by amiloride. ATII cells (greater than 85% pure) were obtained by elastase digestion of lung tissue followed by Percoll centrifugation, and an enriched plasma membrane vesicle fraction was obtained by differential centrifugation. 22Na+ uptake into these vesicles was measured in the presence of a negative inside membrane potential, produced by the addition of the K+ ionophore valinomycin (10 microM) after all external K+ was removed. Electrogenic (valinomycin-sensitive) Na+ uptake (ELNa) was defined as the difference in uptake in the presence and absence of valinomycin. ELNa, normalized per milligram protein, was twice as high across ATII cells than alveolar macrophage membrane vesicles, was inhibited by amiloride (50% inhibitory concentration = 10 microM), and was decreased in the presence of an outwardly directed proton gradient (pHin 6.8; pHout 7.8), suggesting that it was not mediated by Na(+)-H+ antiport. Furthermore, ELNa was equally inhibited by increasing concentrations of amiloride and benzamil but was more sensitive to 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride in concentrations of 10–1,000 microM. These findings indicate that a fraction of Na+ transport across ATII membrane vesicles occurs through a conductive pathway, probably a channel, that has different sensitivity to amiloride and its analogues than the previously described epithelial high amiloride-affinity Na+ channel.

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 MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties.

1987 ◽  
Vol 105 (3) ◽  
pp. 1273-1282 ◽  
Author(s):  
B M Paschal ◽  
H S Shpetner ◽  
R B Vallee
Keyword(s):  
Atpase Activity ◽  
Heavy Chain ◽  
Sedimentation Coefficient ◽  
Minor Component ◽  
High Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Axonemal Dynein ◽  
A Minor

We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.

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