[16] Measurement of Na+ and K+ transport and Na+,K+-ATPase activity in inside-out vesicles from mammalian erythrocytes

1988 ◽  
pp. 171-178 ◽  
Author(s):  
Rhoda Blostein
Keyword(s):  
Atpase Activity ◽  
Inside Out ◽  
K Transport

Membrane domain localization of pH-regulating transporters in frog skeletal muscle membrane vesicles

1996 ◽  
Vol 271 (4) ◽  
pp. C1367-C1379 ◽  
Author(s):  
R. W. Putnam ◽  
P. B. Douglas ◽  
N. A. Ritucci
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Surface Membrane ◽  
Membrane Vesicles ◽  
Muscle Membrane ◽  
Frog Skeletal Muscle ◽  
Membrane Domain ◽  
Ph Response ◽  
Kinase C ◽  
Inside Out

The distribution of pH-regulating transporters in surface and transverse (T) tubular membrane (TTM) domains of frog skeletal muscle was studied. 2',7'-Bis(carboxyethyl)-5(6)- carboxyfluorescein-loaded giant sarcolemmal vesicles, containing surface membrane, exhibited reversible Na+/H+ exchange. A microsomal vesicle fraction was shown to be enriched in TTM on the basis of high Na(+)-K(+)-ATPase and Mg(2+)-ATPase activity, high ouabain and nitrendipine binding, and low Ca(2+)-ATPase activity. TTM vesicles were well sealed and oriented inside out. Vesicles were loaded with the pH-sensitive dye pyranine. In response to an inwardly directed Na+ gradient, vesicles displayed virtually no alkalinization unless monensin was present. No pH response to an imposed Na+ gradient was seen regardless of the direction of the pH gradient across the vesicles, after phosphorylation of the vesicles with protein kinase C, or when exposed to guanosine 5'-O-(3-thiotriphosphate). In the presence of CO2, addition of Na+ or Cl- had no effect on vesicle pH. These data indicate that the TTM lacks functional pH-regulating transporters [Na+/H+ and (Na+ + HCO3-)/Cl- exchangers], suggesting that pH-regulating transporters are localized only to the surface membrane domain in frog muscle.

Mercury inhibits Na-K-ATPase primarily at the cytoplasmic side

1992 ◽  
Vol 262 (5) ◽  
pp. F843-F848 ◽  
Author(s):  
B. M. Anner ◽  
M. Moosmayer
Keyword(s):  
Active Transport ◽  
Metal Binding ◽  
Binding Interface ◽  
Intracellular Proteins ◽  
Active Ion Transport ◽  
A Cell ◽  
Intracellular Domains ◽  
Inside Out ◽  
K Transport ◽  
Cytoplasmic Side

The investigation of active Na-K transport inhibition by mercury is difficult to perform in a cell because of the presence of numerous other membrane and intracellular proteins modifiable by mercury. Thus purified Na-K-adenosinetriphosphatase (ATPase) molecules performing active transport in an artificial membrane are required to demonstrate unequivocally the inhibition of active transport by mercury. We made use of a single population of Na-K-ATPase liposomes filled with ATP and Na to show mercury inhibition of active 86Rb transport mediated by both the inside-out and right-side-out pumps in the same liposome. The effect of HgCl2 on the Na-K-ATPase in cell-like and reversed orientation was measured in comparison with convallatoxin. A dilution series showed that 10 microM externally added HgCl2 inhibited the active 86Rb transport at the cytoplasmic side first; at 50 microM both pump populations were blocked, indicating either membrane permeation by HgCl2 and inhibition at the internal intracellular domains or onset of extracellular action at higher HgCl2 concentration. The results show that the metal-binding interface of Na-K-ATPase molecule is profoundly implicated in active ion transport and that the intracellular part of the Na-K-ATPase molecule presents the primary target for mercury action.

scholarly journals The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme

1993 ◽  
Vol 293 (2) ◽  
pp. 369-375 ◽  
Author(s):  
F L González Flecha ◽  
P R Castello ◽  
A J Caride ◽  
J J Gagliardino ◽  
J P Rossi
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Inorganic Phosphate ◽  
Deleterious Effect ◽  
Kinetic Properties ◽  
Control Activity ◽  
End Products ◽  
Effect Of Glucose ◽  
Inside Out

In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein.

scholarly journals Evidence for High-Affinity Ca2+-ATPase Activity and ATP-Driven Ca2+-Transport in Membrane Preparations of the Gill Epithelium of the Cichlid Fish Oreochromis Mossambicus

1985 ◽  
Vol 119 (1) ◽  
pp. 335-347 ◽  
Author(s):  
GERT FLIK ◽  
JEANNE H. VAN RIJS ◽  
SJOERD E. WENDELAAR BONGA
Keyword(s):  
Atpase Activity ◽  
Plasma Membranes ◽  
Cichlid Fish ◽  
Transport Activity ◽  
High Affinity ◽  
Vesicle Preparation ◽  
Extrusion Mechanism ◽  
Branchial Epithelium ◽  
Inside Out

A high-affinity Ca2+-ATPase activity was demonstrated among the phosphatase activities in plasma membranes of tilapia branchial epithelium: its characteristics (K0.5 = 0.063 μmol l−1 Ca2+, Vmax = 6.02 -mol P1h−;1 mg−1 protein at 37°C) resemble those of Ca2+-translocating enzymes present in mammalian erythrocytes or enterocytes. The ratio of this Ca2+-ATPase activity to Na+/K+-ATPase activity was 1:20.4. Radioimmunoassayable calmodulin was demonstrated in the Ca2+-ATPase-containing membrane fraction. ATP-dependent Ca2+-transport was demonstrated in tight-vesicle preparations of the branchial cell membranes; 30 % of the vesicles in the preparation were inside-out, 44 % were right-side-out and 26 % were leaky. The characteristics of the active Ca2+-transport activity are consistent with a Ca2+-extrusion mechanism involving high-affinity Ca2+-ATPase activity. The branchial Ca2+-transport activity per fish, as calculated on the basis of the transport activity determined for the vesicle preparation, is of the order of the branchial Ca2+-influx rates observed in vivo. The data provide the first biochemical evidence for active Ca2+ -transport in plasma membranes of branchial epithelium. A model is presented for the mechanism of active transepithelial Ca2+-transport in fish gills.

Effect of high extracellular K+ on Na-K-ATPase in cultured canine kidney cells

1990 ◽  
Vol 259 (2) ◽  
pp. F227-F232 ◽  
Author(s):  
M. A. Manuli ◽  
I. S. Edelman
Keyword(s):  
Atpase Activity ◽  
Northern Analysis ◽  
Beta Subunits ◽  
Cell Monolayers ◽  
High K ◽  
Confluent Cell ◽  
K Transport ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Specific Mrna

The Madin-Darby canine kidney (MDCK) cell line was used to evaluate the influence of high extracellular K+, independent of hormonal effects, on renal Na-K-adenosinetriphosphatase (ATPase) activity and abundance. Confluent cell monolayers were incubated in control (5 mM) or high K+ (7.5 mM) medium for 24 h. Exposure to high K+ elicited a 46% rise in Na-K-ATPase activity and a 55% increase in ouabain-sensitive 86Rb uptake. Na-K-ATPase abundance, estimated from the number of ouabain-binding sites, also increased 63% over control in cells exposed to 7.5 mM K+, and as a consequence there was no statistically significant change in the catalytic turnover number. Northern blot analysis using rat cDNA probes for the alpha 1- and beta-subunits showed no corresponding changes in subunit-specific mRNA abundances at 24 h. We conclude that chronic exposure to high extracellular K+ produces a rise in renal epithelial Na-K-ATPase activity and active K+ transport, independent of changes in aldosterone, renal blood flow, or extracellular Na+ concentration. This effect is due to an increase in enzyme abundance rather than a change in catalytic turnover rate. The results of Northern analysis suggest that regulation of Na-K-ATPase activity and abundance by high K+ may involve translational or posttranslational mechanisms, but further study with cDNA probes of canine origin is needed to resolve this issue.

Mitochondrial injury: an early event in cisplatin toxicity to renal proximal tubules

1990 ◽  
Vol 258 (5) ◽  
pp. F1181-F1187 ◽  
Author(s):  
H. R. Brady ◽  
B. C. Kone ◽  
M. E. Stromski ◽  
M. L. Zeidel ◽  
G. Giebisch ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Proximal Tubule ◽  
Threshold Concentration ◽  
Early Event ◽  
Intact Cells ◽  
Mitochondrial Injury ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Early Effects ◽  
K Transport

Oxygen consumption (QO2) and net K+ transport were studied in rabbit proximal tubule suspensions to define the early effects of cisplatin on proximal tubule function. Cisplatin caused dose-dependent inhibition of QO2, which was delayed in onset. The concentration of cisplatin required for inhibition decreased as the duration of exposure was increased [40-min exposure, threshold concentration of 10(-4) M, inhibitor constant (Ki) of 10(-3) M; 4-h exposure, threshold concentration of 3 X 10(-5) M, Ki of 10(-4) M]. Both ouabain-sensitive and ouabain-insensitive QO2 were reduced, indicating inhibition of all adenosinetriphosphatases, including Na(+)- K(+)-ATPase activity. There was a parallel fall in ouabain-sensitive net K+ transport and cytosolic K+ content, confirming the latter observation. Na(+)-K(+)-ATPase activity was unchanged in cell membranes prepared by hypotonic lysis from cisplatin-treated tubules, indicating an indirect cytosol-dependent mechanism of enzyme inhibition. Nystatin-stimulated QO2 was reduced in cisplatin-treated tubules, excluding inhibition of Na+ entry as the mechanism of injury and suggesting mitochondrial injury. The latter was confirmed by measurement of carbonylcyanide-m-chlorophenylhydrazone (CCCP)-uncoupled QO2 in intact cells and ADP-stimulated (state 3) QO2 in digitonin-permeabilized tubules. Furthermore, by maximally stimulating mitochondrial respiration with CCCP and nystatin, it was possible to demonstrate mitochondrial injury at a time when basal QO2 and K+ transport were apparently normal. These data suggest that mitochondrial injury is a central event in cisplatin toxicity to the proximal tubule.

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