Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

Regulation of gastric acid secretion via modulation of a chloride conductance

When gastric microsomes were purified from resting and stimulated rabbit mucosae, they were found to be generally similar in (H+ + K+)-ATPase activity, peptide composition in single-dimension sodium dodecyl sulfate-gel electrophoresis, and in size. In the stimulated vesicles, optimal proton transport activity was found at pH 7.4, 20-50 mM KCl, and 1 mM ATP-Mg. However, in the case of resting vesicles, the presence of valinomycin and an inward Cl-gradient was also necessary for Mg-ATP-dependent proton transport. Measurement of K+ and Cl-diffusion potentials using 3,3-dipropylthiadicarboxocyanine iodide as a potential sensitive dye showed that both resting and stimulated vesicles developed K+ gradient-dependent potentials in the presence of an impermeant anion, but that Cl- gradient-dependent potentials were observed only in the stimulated preparation. 86Rb+ self-exchange was found in both types of vesicles, but Cl- self-exchange was confined to vesicles derived from stimulated mucosae. Putative inhibitors of anion conductance such as furosemide and anthracene 9-carboxylic acid blocked proton transport, Cl- conductance, 36Cl- uptake, and Cl- exchange. The inhibition of proton transport was overcome by valinomycin. ATPase activity in the presence of nigericin, an H+:K+ exchanger, was unaffected by these inhibitors. K+ conductance, Rb+ uptake, and Rb+ exchange were insensitive to these inhibitors. Thus, activation of acid secretion by the stimulated parietal cell appears to involve at least the appearance of a discrete Cl- conductance in the pump-associated membrane.     

Preparation of rat gastric heavy and light microsomal membranes enriched in (H+-K+)-ATPase using 2H2O and Percoll gradients

Gastric heavy microsomal membranes highly enriched in (H+-K+)-ATPase were obtained from cimetidine- or carbachol-treated rats through 2H2O and Percoll gradient centrifugations. Both the resting (cimetidine-treated) and the stimulated (carbachol-treated) heavy membranes which presumably represent the apical membrane of gastric parietal cells were enriched with the polypeptides of 81,000 and 45,000 besides that of 93,000 representing (H+-K+)-ATPase. No apparent differences could be detected between the resting and the stimulated heavy membranes in their polypeptide profiles or their specific activity of (H+-K+)-ATPase. Nevertheless, the level of 86RbCl uptake was greater in the stimulated than the resting heavy microsomal membrane vesicles. The light gastric microsomes which abound in intracellular tubulovesicles containing reserve (H+-K+)-ATPase as isolated from cimetidine-treated rats were similarly purified with respect to (H+-K+)-ATPase. The purified light gastric membranes were largely devoid of the polypeptides of 81,000 and 45,000 found in the heavy gastric membranes. These observations further support the current hypothesis that secretagogues bring about changes in the environment of (H+-K+)-ATPase and induce KCl permeability in the apical membrane of the parietal cells, although at present we have been unable to identify the polypeptide(s) responsible for the KCl pathway.     

Role of membrane-associated thiol groups in the functional regulation of gastric microsomal (H+ + K+)-transporting ATPase system.

The distribution of free thiol groups associated with the membrane proteins of the purified pig gastric microsomal vesicles was quantified, and the relation of thiol groups to the function of the gastric (H+ + K+)-transporting ATPase system was investigated. Two different thiol-specific agents, carboxypyridine disulphide (CPDS) and N-(1-naphthyl)maleimide (NNM) were used for the study. The structure-function relationship of the membrane thiol groups was studied after modification by the probes under various conditions, relating the inhibition of the (H+ + K+)-transporting ATPase to the ATP-dependent H+ accumulation by the gastric microsomal vesicles. On the basis of the extent of stimulation of the microsomal (H+ + K+)-transporting ATPase in the presence and absence of valinomycin (val) about 85% of the vesicles were found to be intact. CPDS at 1 mM completely inhibits the valinomycin-stimulated ATPase and the associated p-nitrophenyl phosphatase with a concomitant inhibition of vesicular H+ uptake. Both the enzyme and dye-uptake activities were fully protected against CPDS inhibition when the treatment with CPDS was carried out in the presence of ATP. ATP also offered protection (about 65%) against NNM inhibition of the (H+ + K+)-transporting ATPase system and vesicular H+ uptake. Under similar conditions ATP also protected about 10 and 6 nmol of thiol groups/mg of protein respectively from CPDS and NNM reaction. Our data suggest that the thiol groups on the outer surface of the vesicles are primarily involved in gastric (H+ + K+)-transporting ATPase function. Furthermore, at least about 15% of the total microsomal thiol groups appear to be associated with the ATPase system. The data have been discussed in terms of the structure-function relationship of gastric microsomes.     

A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.     

Role of cholesterol in the structure and function of gastric microsomal vesicles

Digitonin was used as a tool to investigate the organization and function of cholesterol in gastric microsomes. Microsomal vesicles were treated with digitonin for different time at 0-4 degrees C under isotonic conditions. The effects of digitonin treatment of the vesicles on removal of cholesterol, ultrastructural changes, (H+ + K+)-ATPase activity, and gastric ATPase-dependent H+ uptake ability were investigated. Microsomal cholesterol was extracted in an exponential manner with a t1/2 of 32 min. There was no release of microsomal phospholipids by digitonin treatment during the same period. Digitonin treatment (30 min) produced visible "holes" in the vesicles; at the same time (H+ + K+)-ATPase-dependent H+ uptake was abolished. Under the same conditions the K+-stimulated ATPase activity, however, was moderately (about 35%) reduced, although the response of K+ stimulation to valinomycin was obliterated. Longer digitonin treatment resulted in gradual diffusion and eventual disappearance of the "holes" with the generation of distorted cup-shaped microsomes. The data strongly suggest that membrane lipids are freely mobile and that there is a certain degree of specialization in the organization of gastric microsomal cholesterol for the proper maintenance of the membrane structure and function.     

A study of H+ transport in gastric microsomal vesicles using fluorescent probes.

Fluorescent amines, 9-aminoacridine, acridine orange and quinacrine, were used as probes for a pH gradient (deltapH) across gastric microsomal vesicles. Analysis of probe uptake data indicates that 9-aminoacridine distributes across the membrane as a weak base in accordance with the deltapH. On the other hand, acridine orange and quinacrine show characteristics of binding to membrane sites in addition to the accumulation in response to deltapH. A discussion of the advantages and limitations of the probes is presented. Application of these probes to pig gastric microsomal vesicles indicates that that K+-stimulated ATPase is responsible for the transport of H+ into the vesicles and thus develops a deltapH across the membrane. The deltapH generated by the K+-ATPase has a definite requirement for internal K+. The proton gradient can be discharged slowly after ATP depletion or rapidly either by detergent disruption of the vesicles or by increasing their leakiness using both H+ and K+ ionophores. On the other hand, the sole use of the K+ ionophore, valinomycin, stimulates the ATP-induced formation of deltapH by increasing the availability of K+ to internal sites. This stimulation by valinomycin requires the presence of permeable anions like Cl-. Analysis of the Cl- requirement indicates that in the presence of valinomycin the net effect is the accumulation of HCl inside the gastric vesicles. With an external pH of 7.0, the ATP-generated deltapH was calculated to be from 4 to 4.5 pH units. The results are consistent with the hypothesis that the K+-stimulated ATPase drives a K+/H+ exchange across the gastric vesicles. Since other lines of evidence suggest that these gastric microsomes are derived from the tubulovesicular system of the oxyntic cell, the participation of the ATP-driven transport processes in gastric HCl secretion is of interest.     

Finding of a KCl-independent, electrogenic, and ATP-driven H+-pumping activity in rat light gastric membranes and its effect on the membrane K+ transport activity

Resting rat light gastric membranes prepared through 2H2O and Percoll gradient centrifugations were enriched not only with (H+-K+)-ATPase and K+ transport activity (Im, W. B., Blakeman, D. P., and Davis, J. P. (1985) J. Biol. Chem. 260, 9452-9460), but also with a K+-independent, ATP-dependent H+-pumping activity. This intravesicular acidification has been ascribed to an oligomycin-insensitive H+-ATPase which differed from (H+-K+)-ATPase in several respects. The H+-ATPase is electrogenic, apparently of lower capacity, required a lower optimal ATP concentration (4 microM for the H+-ATPase and 500 microM for (H+-K+)-ATPase), of lower sensitivity to vanadate and sulfhydryl agents such as p-chloromercuribenzoate and N-ethylmaleimide, and insensitive to SCH 28,080, a known competitive inhibitor of (H+-K+)-ATPase with respect to K+. Operation of the H+-ATPase, however, appeared to interfere with the K+ transport activity in the light gastric membranes, probably through development of intravesicular positive membrane potential; for example, micromolar levels of Mg2+-ATP fully inhibited K+ uptake and stimulated K+ efflux as measured with 86Rb+. Involvement of (H+-K+)-ATPase in the K+ transport is not likely, since the inhibitory effect of Mg2+-ATP continued even after removal of the nucleotide with an ATP-scavenging system. Moreover, nigericin, an electroneutral H+/K+ exchanger, could bypass the inhibitory effect of Mg2+-ATP and equilibrate the membrane vesicles with 86Rb+ while valinomycin, an electrogenic K+ ionophore, could not. Finally, the H+-ATPase could possibly be involved in the acid secretory process, since its H+-pumping activity was removed from the light gastric membrane fraction upon carbachol treatment, along with the K+ transport and (H+-K+)-ATPase activities. We have speculated that the H+-ATPase is responsible for maintaining the K+-permeable intracellular membrane vesicles acidic and K+ free during the resting state of acid secretion and may contribute to basal acid secretion.     

Cation transport by gastric H+:K+ ATPase.

A vesicular microsomal fraction isolated from hog fundic mucosa demonstrates the capacity to take up equal amounts of RB+ and Cl-. The amount of the Rb+ uptake is sensitive to the extravesicular osmolarity, and rate of uptake is sensitive to temperature. 86Rb+ efflux is dependent upon the cation composition of the diluting solution. ATP, but not beta-gamma methylene ATP, induces a reversible efflux of 86Rb+ from loaded vesicles, and this is dependent upon a functional K+-ATPase. The ATP induced efflux is not affected by CCCP (carbonyl cyanide m-chlorophenylhydrazone) or TCS (tetrachlorosalicylanilide) nor by lipid soluble ions or valinomycin. Nigericin inhibits the efflux by 40%. Uptake of the lipid soluble ion 14C-SCN- has been demonstrated and is enhanced by ATP only in the presence of valinomycin. The results are consistent with a neutral or isopotential exchange of H+ for Rb+ mediated by K+-ATPase.     

Correlation between microsomal (Na+ + K+)-ATPase activity and [3H]ouabain binding to heart tissue homogenates.

ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples.     

Inhibition of gastric (H+ + K+)-ATPase by the substituted benzimidazole, picoprazole

The substituted benzimidazole, picoprazole, inhibited the gastric (H+ + K+)-ATPase in a concentration-and time-dependent manner. Half-maximal inhibition of the (H+ + K+)-ATPase activity was obtained at about 2 . 10(-6)M under standard conditions. In addition to the inhibition of ATPase activity, parallel inhibition of phosphoenzyme formation and the proton transport activity were achieved. Radiolabelled picoprazole was found to bind to 100 kDa peptide; this peptide was shown by phosphorylation experiments to contain the catalytic centre of the (H+ + K+)-ATPase. Studies on the (Na+ + K+)-ATPase indicated that this enzyme was unaffected by picoprazole. From the data presented and from other pharmacological studies, it is proposed that this compound inhibits acid secretion at the level of the parietal cell by its ability to inhibit the gastric proton pump, the (H+ + K+)-ATPase.     

Inhibition of (H+ + K+)-ATPase by omeprazole in isolated gastric vesicles requires proton transport

Omeprazole was found to inhibit the (H+ + K+)-ATPase activity in isolated gastric vesicles only when acid was accumulated in the vesicle lumen. The ATPase activity was time- and dose-dependently inhibited in the presence of K+ and valinomycin. Under conditions in which no pH-gradient was generated, i.e., in the presence of K+ alone or NH4+, no effect of omeprazole was found. The degree of inhibition was directly correlated to the amount of inhibitor bound to the preparation. A stoichiometry of 2 mol radiolabelled inhibitor bound per mol phosphoenzyme was found on total inhibition of the K+ plus valinomycin-stimulated activity. This inhibitory action of omeprazole on the ATPase activity could be fully reversed by addition of beta-mercaptoethanol. The inhibition of the proton transport in the (H+ + K+)-ATPase-containing vesicles by omeprazole was also strictly correlated to the amount of bound inhibitor. The stoichiometry of binding at total inhibition of this reaction was found to be 1.4 mol per mol phosphoenzyme. The K+-stimulated p-nitrophenylphosphatase activity was inhibited in parallel with the ATPase activity, whereas the phosphoenzyme levels were affected to a lesser extent by omeprazole. Gel electrophoresis of an omeprazole-inhibited vesicle preparation showed that the radiolabel was mainly found at 94 kDa, the molecular weight of the (H+ + K+)-ATPase catalytic subunit(s).     

Interaction of anions and ATP with the coated vesicle proton pump

ATP-driven proton transport in intact clathrin-coated vesicles requires the presence of a permeant anion, such as Cl-, to provide charge compensation during the electrogenic movement of protons. Using the purified (H+)-ATPase from clathrin-coated vesicles in both the detergent-solubilized and reconstituted states, we have studied the direct effects of anions on the activity of this enzyme. Both proton transport and ATP hydrolysis by the purified enzyme are independent of the presence of Cl-. In addition, proton transport does not occur even at high Cl- concentrations unless K+ and valinomycin are present to dissipate the membrane potential generated. These results indicate that the anion channel which provides for Cl- flux in intact coated vesicles is not a component of the purified (H+)-ATPase. Inhibition of ATPase activity is observed in the presence of I-, NO3-, or SO4(2-), with 50% inhibition occurring at 350 mM I-, 50 mM NO3-, or 40 mM SO4(2-). The presence of ATP lowers the concentration of I- required for 50% inhibition from 350 mM to 100 mM and increases the maximal inhibition observed in the presence of NO3- from 65% to 100%. Two separate mechanisms appear to be responsible for anion inhibition of the (H+)-ATPase. Thus, I- and high concentrations of NO3- (in the presence of ATP) cause inhibition by dissociation of the (H+)-ATPase complex, while SO4(2-) and NO3- (in the absence of ATP) cause inhibition without dissociation of the complex, suggesting the existence of an inhibitory anion binding site on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla

When purified on a sucrose gradient, basolateral membranes from dog kidney outer medulla are found to be very rich in (Na,K)-ATPase; about 50% of the membrane protein is comprised of this enzyme. (Na,K)-ATPase activity is activated 3- to 5-fold by detergent treatment, and this has been previously attributed to the impermeable vesicular nature of the membranes. Porcine trypsin inactivates only that fraction of (Na,K)-ATPase activity seen without detergent, consistent with a right-side-out orientation of membrane vesicles; the trypsin sensitivity and detergent activation of [3H]ouabain binding in the presence of Na+ + Mg2+ + ATP or Mg2+ + Pi are also consistent with this hypothesis. Using nearly isosmotic Hypaque density gradient centrifugation a population of impermeable right-side-out membrane vesicles (H1) is separated from a leaky population (H2). (Na,K)-ATPase activity in the H1 population is 20-fold activated by detergent and insensitive to porcine trypsin. The vesicle volume is 2.4 microliters/mg, and monovalent cations passively equilibrate with the intravesicular volume on a time scale of 5-30 min. Very rapid ouabain sensitive 22Na efflux from the vesicles is observed when ATP is photolytically released from intravesicular caged ATP.     

Inhibition by lead ion of Electrophorus electroplax (Na+ + K+)-adenosine triphosphatase and K+-p-nitrophenylphosphatase.

Inorganic lead ion in micromolar concentrations inhibits Electrophorus electroplax microsomal (Na+ + K+)-adenosine triphosphatase ((Na+ + K+)-ATPase) and K+-p-nitrophenylphosphatase (NPPase). Under the same conditions, the same concentrations of PbCl2 that inhibit ATPase activity also stimulate the phosphorylation of electroplax microsomes in the absence of added Na+. Enzyme activity is protected from inhibition by increasing concentrations of microsomes, ATP, and other metal ion chelators. The kinetics follow the pattern of a reversible noncompetitive inhibitor. No kinetic evidence is elicited for interactions of Pb2+ with Na+, K+, Mg2+, ATP, or p-nitrophenylphosphate. Na+- ATPase, in the absence of K+, and (Na+ + K+)-NPPase activity at low [K+] are also inhibited. ATP inhibition of NPPase is not reversed by Pb2+. The calculated concentrations of free [Pb2+] that produce 50% inhibition are similar for ATPase and NPPase activities. Pb2+ may act at a single independent binding site to produce both stimulation of the kinase and inhibition of the phosphatase activities.     

Changes in the membrane environment of the (K+ + H+)-ATPase following stimulation of the gastric oxyntic cell

Biochemical evidence is presented for changes in the membrane environment of the (K+ + H+)-dependent ATPase enzyme of the oxyntic cell following in vivo gastric stimulation of young New Zealand rabbits. The changes are inferred from the marked differences in the sedimentation properties of the (K+ + H+)-ATPase when obtained from homogenates of either stimulated or nonstimulated (resting) fundic gastric epithelium. Stimulation resulted in a redistribution of K+-ATPase activity that was reduced to less than half in the microsomal pellet and concomitantly increased in the membrane fractions normally associated with nuclei and mitochondria. Density gradient fractionation of the mitochondrial pellet yield a preparation rich in (K+ + H+)-ATPase. Our studies indicated that the membranes in this preparation are far larger and apparently denser than the microsomal vesicles associated with the nonstimulated state of the cell. The specific nature of the relationship between stimulation and the observed changes is suggested by the lack of change in the distribution of enzymatic activities unrelated to the apical pole of the oxyntic cell. Preliminary, tentative information aimed at identifying the processes responsible for the observed changes is presented.     

Inhibition of (H+ + K+)-ATPase and H+ accumulation in hog gastric membranes by trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate

The mechanism of gastric antisecretory action for trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) has been studied utilizing isolated hog gastric membranes enriched with (H+ + K+)-ATPase. The drugs inhibited the gastric ATPase due to their apparent competition with K+ for the luminal high-affinity K+-site of the ATPase. The dose to inhibit 50% (ID50) of the ATPase in the membranes rendered freely permeable to K+ (20 mM) was 50 microM for trifluoperazine and 1.5 mM for verapamil and TMB-8. In intact hog gastric membranes which develop a pH gradient in the presence of valinomycin, ATP and KCl, however, trifluoperazine at 4 microM, verapamil and TMB-8 at 15 microM inhibited 40 and 30% of the valinomycin-stimulated ATPase activity, respectively, and also blocked the ionophore-dependent intravesicular acidification as measured by aminopyrine accumulation. The enhanced potency of the drugs to inhibit the ATPase in the intact membrane vesicles may be attributed to the accumulation of the drugs as a weak base within the vesicles, where the luminal K+-site of the ATPase is accessible. Calmodulin and Ca2+ had no effect on the extent of H+-accumulation as measured by aminopyrine accumulation in the membrane vesicles which were prepared in the presence of 1 mM EGTA. Since the drugs showed similar potency in interfering with H+ movements either in the membrane vesicles or isolated rabbit gastric glands stimulated by dibutyryl cAMP, it is reasonable to suggest the inhibitory effect of the drugs on (H+ + K+)-ATPase as a primary cause for such interferences in both cases. A trifluoperazine analog and other lipophilic amine drugs similarly inhibited (H+ + K+)-ATPase and H+ accumulation in the membrane vesicles or in the glands. We have concluded that a tertiary amine, the only common functional group among these drugs, is primarily responsible for their ability to interact with the high-affinity K+ site of the gastric ATPase.     

A protein phosphatase associated with rat heavy gastric membranes enriched with (H+-K+)-ATPase influences membrane K+ transport activity

Rat stimulated heavy gastric membranes enriched with (H+-K+)-ATPase, a marker for the apical membrane of the parietal cell, displayed a 32P-histone-dephosphorylating activity which appeared to be physically copurified with, but functionally independent of, the ATPase. The protein phosphatase activity was optimal at pH 7.5 and was inhibited by fluoride (50 mM), inorganic phosphate (50 mM), and p-chloromercuribenzoate (0.1 mM), but was insensitive to vanadate (1 mM). The 32P-phosphoproteins in the heavy gastric membranes were also dephosphorylated, apparently by their own membrane-bound phosphatase in the presence of Mg2+ at millimolar concentrations, which is likely to enhance membrane-membrane interaction. Heavy gastric membrane vesicles incubated with Mg2+ (2 mM) exhibited no alterations in K+-dependent ATP-hydrolyzing activity, Cl permeability, and protein and lipid compositions, but irreversibly lost the ATP, K+-dependent H+-pumping activity. Since valinomycin, a K+-specific ionophore, restored the intravesicular acidifying activity and an inhibitor of the protein phosphatase, inorganic phosphate, largely blocked the Mg2+-induced change in the membrane transport function, it is reasonable to propose that the phosphatase action on certain membrane proteins, possibly the putative K+ transporter or regulatory proteins, selectively decreases K+-conductance in the apical membranes of gastric parietal cells.     

Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles.

The Mg2+-dependent, K+-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H+ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth. Valinomycin stimulated both dye uptake and K+-ATPase (valinomycin-stimulated K+-ATPase); stimulation by valinomycin was due to increased K+ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K+-ATPase and H+ accumulation as an index, the sequence for anion permeation was NO-3 greater than Br- greater than Cl- greater than I- greater than acetate approximately isethionate. When permeability to both K+ and H+ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K+-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an 'uncoupling' of ATP hydrolysis from H+ accumulation. Thiocyanate interacts with valinomycin inhibiting the typical action of the K+ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN- to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K+-ATPase, it appears that SCN- is a very permeant anion which can be placed before NO-3 in the sequence of permeation. Valinomycin-stimulated ATPase and H+ uptake showed similar dependent correlations, including: dependence on [ATP] and [K+], pH optima, temperature activation, and selective inhibition by SH- or NH2-group reagents. These results are consistent with a pump-leak model for the gastric microsomal K+-ATPase which was simulated using Nernst-Planck conditions for passive pathways and simple kinetics for the pump. The pump is a K+/H+ exchange pump requiring K+ at an internal site. Rate of K+ entry would depend on permeability to K+ as well as the counterion, either (1) the anion to accompany K+ or (2) the H+ efflux path as an exchange ion. The former leads to net accumulation of H+ and anion, while the latter results in non-productive stimulation of ATP hydrolysis.     

(Na+ + K+)-ATPase in artificial lipid vesicles: influence of the concentration of mono- and divalent cations on the pumping rate

(Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. Transport activity was induced by adding ATP to the external medium. A voltage-sensitive dye was used to detect the ATP-driven potassium extrusion in the presence of valinomycin. The observed substrate-protein interactions of the reconstituted (Na+ + K+)-ATPase largely agree with that from native tissues. An agreement between ATP hydrolysis and transport activity is given for concentration dependences of sodium, potassium, magnesium and calcium ions. The only significant deviations were observed in the influence of pH. Protons were found to have different influence on transport, enzymatic activity and phosphorylation of the enzyme. The transport studies showed a twofold interaction of protons with the protein: competition with sodium at the cytoplasmic ion binding sites, a non competitive inhibition of transport which is not correlated with protein phosphorylation.