Proton/hydroxyl permeability of proximal tubule brush border vesicles

1985 ◽  
Vol 248 (1) ◽  
pp. F78-F86 ◽  
Author(s):  
H. E. Ives
Keyword(s):  
Activation Energy ◽  
Proximal Tubule ◽  
Brush Border ◽  
Buffer Capacity ◽  
Gradient Centrifugation ◽  
Sulfhydryl Group ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Gradients

The net H+/OH- permeability of rabbit renal proximal tubule brush border membrane vesicles was determined by measuring the rate of collapse of preformed pH gradients using acridine orange. The membranes were voltage clamped using valinomycin and [K+]in = [K+]out. Internal buffer capacity was determined by titration of lysed vesicles and by titration of measured Na+/H+ exchange rates with exogenously added buffers. Both methods revealed an intravesicular buffer capacity of 125-135 mM/pH unit at pH 6.0 and 20 degrees C. Using this buffer capacity, the net H+/OH- permeability was found to be 5 X 10(-3) cm/s in brush border vesicles prepared by Mg2+ aggregation. The rate of collapse of pH gradients in brush border vesicles prepared by sucrose density gradient centrifugation was virtually identical to the rate in vesicles prepared with Mg2+, indicating that the high H+/OH- permeability was not an artifact of Mg2+ preparation. Activation energy of the H+/OH- permeability pathway was 4.9 kcal/mol, whereas activation energy of the Na+/H+ antiporter was 11.4 kcal/mol. Since the rate of H+/OH- diffusion was not affected by amiloride, it is concluded that H+/OH- permeate through brush border membranes by a pathway separate from the Na+/H+ antiporter. This pathway is not inhibited by dicyclohexylcarbodiimide at concentrations up to 2 mM but is inhibited by 0.2-5 mM p-chloromercuribenzenesulfonate, suggesting the presence of a sulfhydryl group in the pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

1973 ◽  
Vol 58 (2) ◽  
pp. 436-462 ◽  
Author(s):  
Gert Kreibich ◽  
Pascale Debey ◽  
David D. Sabatini
Keyword(s):  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Transport Systems ◽  
Secretory Proteins ◽  
Sucrose Density Gradient Centrifugation ◽  
Detergent Concentration ◽  
Doc Concentration ◽  
Microsomal Membranes

Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025–0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.

Na+/H+ exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells

2002 ◽  
Vol 283 (3) ◽  
pp. C927-C940 ◽  
Author(s):  
S. Akhter ◽  
O. Kovbasnjuk ◽  
X. Li ◽  
M. Cavet ◽  
J. Noel ◽  
...  
Keyword(s):  
Cell Surface ◽  
Proximal Tubule ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Insoluble Fraction ◽  
Proximal Tubule Cell ◽  
Apical Surface ◽  
Sucrose Density Gradient Centrifugation ◽  
Opossum Kidney ◽  
Ok Cells

Cell biological approaches were used to examine the location and function of the brush border (BB) Na+/H+ exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10–15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as ∼400- and ∼900-kDa standards, whereas surface NHE3V was enriched in the ∼900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from ∼400 to ∼900 kDa.

scholarly journals Plasma Membrane Vesicles from Mouse 15091A Tumor Cells: Agent of Platelet Aggregating Activity

1977 ◽  
Author(s):  
G.J. Gasic ◽  
J.L. Catafalmo ◽  
G.P. Gasic ◽  
S.J. Shattil ◽  
G.J. Stewart
Keyword(s):  
Plasma Membrane ◽  
Tumor Cells ◽  
Cell Transformation ◽  
Gradient Centrifugation ◽  
Amorphous Material ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicles ◽  
Sucrose Density Gradient Centrifugation ◽  
Artificial Plasma

We reported that cells from most tumors display platelet aggregating activity (PAA) in heparinized plasma and that this activity contributes to metastasis.Recently, we demonstrated that PAA can be used as a marker of cell transformation in virally infected rat cells. The material responsible for PAA is shed into culture medium. Characterization revealed a material which is particulate and sedimentable at 50,000x g for 60 min.; it contains proteins and lipids with a free cholesterol to phospholipid ratio of 0.556.Delipida-tion as well as complete solubilization abolished PAA.SDS-ME PAGE, 7.5% slab gels, revealed 20 bands.EM studies of 50,000x g pellets shed by 15091A cells indicated they contained numerous vesicles, some solid bodies, numerous free or vesicle associated small particles, and some amorphous material. Discontinuous sucrose density gradient centrifugation of the 50,000x g pellet yielded at the 1.07-1.17 g/cm3 interface a predominantly vesicular fraction which was the most active interfacial material. The vesicles, visible with phase contrast microscopy, resemble those produced by artificial plasma membrane vesiculation in various cell systems, including normal cells. Since PAA is only shown by transformed cells, vesicles from these must be different or much more numerous. Spontaneous vesiculation by tumor cells may be potentially important in understanding cell transformation and tumor metastases.

Parathyroid hormone and dibutyryl cAMP inhibit Na+/H+ exchange in renal brush border vesicles

1985 ◽  
Vol 248 (2) ◽  
pp. F212-F218 ◽  
Author(s):  
A. M. Kahn ◽  
G. M. Dolson ◽  
M. K. Hise ◽  
S. C. Bennett ◽  
E. J. Weinman
Keyword(s):  
Parathyroid Hormone ◽  
Proximal Tubule ◽  
Brush Border ◽  
Sodium Phosphate ◽  
Brush Border Membrane ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Enzymatic Digestion ◽  
Dibutyryl Camp ◽  
Ficoll Gradient

Parathyroid hormone (PTH) and cAMP inhibit sodium, water, and bicarbonate reabsorption in the proximal tubule. We wished to determine whether these agents directly inhibit proximal tubular Na+/H+ exchange. A suspension of rabbit proximal tubules was prepared by enzymatic digestion and Ficoll gradient centrifugation. Oxygen consumption at 37 degrees C was stable over 60 min, averaged 20 nmol X mg protein-1 X min-1, and was inhibited 60% by ouabain. Over 96% of cells excluded trypan blue. From this suspension, brush border membrane vesicles were isolated. The vesicles were enriched 12.7 times in alkaline phosphatase relative to a cortical homogenate and demonstrated pH gradient-stimulated, amiloride-sensitive Na+/H+ countertransport and sodium-phosphate and sodium-D-glucose cotransport. When the tubule suspension was exposed to PTH or dibutyryl cAMP, the activity of Na+/H+ countertransport in the resultant brush border vesicles was inhibited. Neither PTH nor dibutyryl cAMP affected the amiloride-insensitive component of sodium transport or sodium-phosphate or sodium-D-glucose cotransport. The effect of PTH on Na+/H+ counter-transport could not be explained by an alteration in fluidity of the brush border membrane. These experiments demonstrate that PTH and dibutyryl cAMP directly inhibit Na+/H+ countertransport in the brush border membrane of the rabbit proximal tubule.

Galactosyltransferase activity is not localized to the brush border membrane of human small intestine

1986 ◽  
Vol 6 (2) ◽  
pp. 171-175 ◽  
Author(s):  
Frances Boyle ◽  
Susan Snape ◽  
Paul Duane ◽  
Neil Cook ◽  
Timothy Peters
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Equilibrium Density ◽  
Sucrose Density Gradient Centrifugation ◽  
Human Small Intestine ◽  
Immunocytochemical Techniques

A recent report [Roth et al. (1985) J. Cell Biol.100: 118–125], using immunocytochemical techniques, calimed that human duodenal galactosyltransferase is located predominantly on the external aspect of enterocyte brush border membranes. Analytical subcellular fractionation by sucrose density gradient centrifugation of human jejunum biopsy homogenates demonstrated that galactosyltransferase activity is localized to the Golgi fraction (equilibrium density of 1.14 g cm−3) and is not found in significant amounts in the brush border membrane (equilibrium density of 1.22 g cm−3).

scholarly journals Reconstitution of ciliary membranes containing tubulin.

1983 ◽  
Vol 96 (1) ◽  
pp. 68-75 ◽  
Author(s):  
R E Stephens
Keyword(s):  
Gradient Centrifugation ◽  
Protein Composition ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Major Protein ◽  
Sucrose Density Gradient Centrifugation ◽  
Thaw Cycle ◽  
Free Membrane ◽  
Brain Tubulin ◽  
Membrane Tubulin

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

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