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.     

Purification and partial characterization of the (H+,K+)-transporting adenosinetriphosphatase from fundic mucosa

The microsomal (H+,K+)-ATPase systems from dog and pig fundic mucosa were purified to homogeneity and partially characterized. The method involves sodium dodecyl sulfate (SDS) (0.033% w/v) extraction of the microsomal non-ATPase proteins under appropriate conditions followed by sucrose density gradient centrifugation. Two distinct membrane bands of low (buoyant density = 1.08 g/mL) and high (buoyant density = 1.114 g/mL) densities having distinct enzymatic and chemical composition were harvested. The low-density membrane was highly enriched in Mg2+- or Ca2+-stimulated ATPase and 5'-nucleotidase activities but totally devoid of (H+,K+)-ATPase and K+-p-nitrophenylphosphatase activities. The latter two activities were found exclusively in the high-density membrane. SDS-polyacrylamide gel electrophoresis revealed the high-density membranes to consist primarily of a major 100-kilodalton (kDa) protein and a minor 85-kDa glycoprotein, the former being the catalytic subunit of the (H+,K+)-ATPase. The amino acid composition of the pure dog (H+,K+)-ATPase revealed close similarities with that from pig. The N-terminal amino acid was identified to be lysine as the sole residue. Similar to the high-density membrane-associated pure (H+,K+)-ATPase, the low-density membranes containing high Mg2+-ATPase activity also contained a 100-kDa peptide and a 85-kDa glycopeptide in addition to numerous low molecular weight peptides. Also, similar to the pure (H+,K+)-ATPase, the Mg2+-ATPase-rich fraction produced an E approximately P unstable to hydroxylamine and partially (about 25%) sensitive to K+ but having a slow turnover. The levels of E approximately P produced by the pure (H+,K+)-ATPase- and Mg2+-ATPase-rich fractions were 1400 and 178 pmol/mg of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characteristics of a pure endogenous activator of the gastric H+,K+-ATPase system. Evaluation of the role as a possible intracellular regulator

An endogenous activator capable of stimulating the gastric H+,K+-ATPase activity has been purified to homogeneity from dog and pig gastric cells and found to be a dimer of two identical 40-kDa subunits in the active state. Identical nature of the activator monomers was revealed by the detection of lysine as the sole N-terminal amino acid. The activator from one species can stimulate the H+,K+-ATPase from another species and vice versa. Such cross-activation is consistent with the striking similarities in the amino acid composition between the two species, suggesting considerable homology in the activator molecules from different species. The activator exhibited several unique features during modulation of the H+,K+-ATPase reaction. It appreciably enhances affinity of the H+,K+-ATPase for K+, known to increase turnover of the enzyme. To complement this K+ affinity, the activator also enhances ability of the H+,K+-ATPase to generate more transition state (E*.ATP) complex by increasing the entropy of activation (delta S++) of the system as revealed from an Arrhenius plot of the data on temperature activation. In addition, the activator shows both positive cooperativity and strong inhibition, depending on its concentration. Thus, up to the ratio of the H+,K+-ATPase and activator of about 1:2 (on the protein basis), the activator shows sigmoidal activation (Hill coefficient = 4.5), but beyond such concentration a strong inhibition was observed. Finally, Ca2+ at low (2-4 microM) concentration strongly inhibits the activator-stimulated H+,K+-ATPase. It is proposed that the activator may be acting as a link in the signal transducing cascade system between the intracellular second messenger (Ca2+) and the physiological response (gastric H+ transport).     

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

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.     

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.     

Purification and characterization of a cytosolic activator protein for the gastric H+,K+-ATPase system from dog fundic mucosa

An endogenous protein activator (AF) responsible for the activation of the gastric H+,K+-ATPase system, identified recently as the biochemical mechanism for the transport of H+, has been purified to homogeneity and partially characterized. The purification procedure (at 0-4 degrees C) involves simultaneous concentration and dialysis of the cytosolic fraction from dog fundic cells under negative pressure, pH 4.8 precipitation and two consecutive gel filtration steps on sephacryl S-200 columns. The highly purified and active AF is a protein of 80 Kd consisting of two identical subunits of 40 Kd each. The AF not only stimulates the gastric H+,K+-ATPase activity but also greatly enhances the rate of ATPase dependent proton pumping inside gastric microsomal vesicles. The data clearly suggest an important regulatory role of the cytosolic AF in the gastric HCl secretory process.     

Effects of cholesterol on (Na+,K+)-ATPase ATP hydrolyzing activity in bovine kidney

The (Na+,K+)-ATPase ATP hydrolyzing activity from rabbit kidney medulla basolateral membrane vesicles was studied as a function of the cholesterol content of the basolateral membranes. The cholesterol content of the membranes was modified by incubation with phospholipid vesicles. When the cholesterol content was increased above that found in the native membrane, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. When the cholesterol content was decreased from that found in the native membranes, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. Analogous effects were found with the K+-activated phosphatase activity of the same membrane vesicles. Therefore, at low cholesterol contents, cholesterol was stimulatory, and at high cholesterol contents, cholesterol was inhibitory. The structural specificity of this effect was tested by introducing lanosterol and ergosterol as 50% of the membrane sterol. Ergosterol was the least effective at supporting (Na+,K+)-ATPase ATP hydrolyzing activity, while lanosterol was more effective, but still not as effective as cholesterol.     

The unique ultrastructure of secretory membranes in gastric parietal cells depends upon the presence of H+, K+ -ATPase

Ion transporters play a central role in gastric acid secretion. To determine whether some of these transporters are necessary for the normal ultrastructure of secretory membranes in gastric parietal cells, mice lacking transporters for H+, K+, Cl-, and Na+ were examined for alterations in volume density (Vd) of basolateral, apical, tubulovesicular and canalicular membranes, microvillar dimensions, membrane flexibility, and ultrastructure. In mice lacking Na+/H+ exchanger 1 (NHE1) or the Na+-K+-2Cl- cotransporter (NKCC1), the ultrastructure and Vd of secretory membranes and the secretory canalicular to tubulovesicular membrane ratio (SC/TV), a morphological correlate of secretory activity, were similar to those of wild-type mice. In mice lacking Na+/H+ exchanger 2 (NHE2) or gastric H+, K+ -ATPase alpha- or beta-subunits, the SC/TV ratio and Vd of secretory membranes were decreased, though canaliculi were often dilated. In H+, K+ -ATPase-deficient parietal cells, canalicular folds were decreased, normally abundant tubulovesicles were replaced with a few rigid round vesicles, and microvilli were sparse, stiff and short, in contrast to the long and flexible microvilli in wild-type cells. In addition, microvilli of the H+, K+ -ATPase-deficient parietal cells had centrally bundled F-actin filaments, unlike the microvilli of wild-type cells, in which actin filaments were peripherally positioned concentric to the plasmalemma. Data showed that the absence of H+, K+ -ATPase produced fundamental changes in parietal cell membrane ultrastructure, suggesting that the pump provides an essential link between the membranes and F-actin, critical to the gross architecture and suppleness of the secretory membranes.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

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.     

Biosynthesis of glycerolipids by hepatoma and liver microsomes. I. Fatty acyl-CoA ligase and acyl-CoA:sn-glycerol-3-phosphate acyltransferase

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on ouabain-Sepharose. By the method used two subfractions were obtained, one eluting freely from the affinity gel (MF1oua) and a second specifically retained by matrix-bound ouabain (MF2oua), with a total recovery of 95 per cent. Fractionation required the binding of matrix-bound ouabain to its plasma membrane receptor, i.e. (Na+ + K+)-ATPase. Increasing the temperature and binding time did not significantly alter the fractionation of plasma membranes into the two subfractions. Both plasma membrane subfractions separated by ouabain-Sepharose were of plasma membrane origin, as revealed by the identical specific activities of several membrane bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase and Mg2+-ATPase in unseparated plasma membranes and in both subfractions, and by the identical amounts of the cytoskeletal protein actin in unseparated plasma membranes and subfractions. The plasma membrane subfractions MF1oua and MF2oua showed different structural and functional properties. In SDS-polyacrylamide gel electrophoresis polypeptides of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2oua. The phospholipid fatty acid composition of the plasma membrane subfractions proved to be different, as well. MF2oua contained significantly higher amounts of saturated fatty acids as compared to MF1oua. The specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysolecithin acyltransferase were highly enriched in the adherent fraction MF2oua, as compared to MF1oua. The data suggest that by the means of affinity chromatography on ouabain-Sepharose plasma membrane domains of the lymphocyte plasma membrane can be isolated, most probably implicated in the initiation of lymphocyte activation.     

Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells

We studied whether K+-Cl(-) cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H+,K+-ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles. KCC4 coimmunoprecipitated with H+,K+-ATPase in the lysate of SAV. Interestingly the MgATP-dependent uptake of (36)Cl(-) into the SAV was suppressed by either the H+,K+-ATPase inhibitor (SCH28080) or the KCC inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The KCC inhibitor suppressed the H+ uptake into SAV and the H+,K+-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K+-Cl(-) cotransport by KCC4 is tightly coupled with H+/K+ antiport by H+,K+-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H+,K+-ATPase, KCC4 was coimmunoprecipitated with H+,K+-ATPase. The rate of recovery of intracellular pH in the KCC4-expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4-non-expressing cells. Our results suggest that KCC4 and H+,K+-ATPase are the main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.     

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.     

Dog kidney (Na+,K+)-ATPase is more sensitive to inhibition by vanadate than human red cell (Na+,K+)-ATPase

(Na+,K+)-ATPase (EC 3.6.1.3) from kidney is more sensitive to inhibition by vanadate than red cell (Na+,K+)-ATPase. The difference appears to be in the apparent affinities of the two enzymes for K+ and Na+ at sites where K+ promotes and Na+ opposes vanadate binding. As a result of Na+-K+ competition at these sites, reversal of vanadate inhibition was accomplished at lower Na+ concentrations in red cell than in kidney (NA+,K+)-ATPase. It is possible that vanadate could selectively regulate Na+ transport in the kidney.     

Thiophosphoryl guanine nucleotide analogues inhibit the (Na+,K+)-ATPase.

The effects of several guanine nucleotide analogues on (Na+,K+)-ATPase activity of membranes isolated from several tissues were analyzed to determine if a G-protein might be involved in the hormonal regulation of the (Na+,K+)-ATPase. Submillimolar concentrations of GTP gamma S, but not GMPPNP, inhibit rat skeletal muscle and axolemma, but not kidney, (Na+,K+)-ATPase activity. Furthermore, GDP beta S does not reverse GTP gamma S inhibition, but rather itself slightly inhibits (Na+,K+)-ATPase activity. Dithiothreitol can block and reverse GTP gamma S inhibition of skeletal muscle (Na+,K+)-ATPase; the results obtained with axolemma membranes are complicated by the inhibition of (Na+,K+)-ATPase activity in these membranes by DTT. Results showing that high membrane concentrations can mute the inhibitory action of GTP gamma S suggest that a minor contaminant in GTP gamma S preparations is responsible for inhibiting (Na+,K+)-ATPase activity. Neither vanadate, a heavy metal, GDP, phosphate, nor thiophosphate, however, is responsible for this inhibition, and the inhibitory activity elutes with GTP gamma S from Sephadex G-10 columns. It is concluded that GTP gamma S or a structural derivative of GTP gamma S inhibits the (Na+,K+)-ATPase, in a tissue-specific manner, not by interaction with a G-protein as a GTP analogue, but through a direct chemical interaction with the (Na+,K+)-ATPase or some regulatory protein. The terminal SH group of the nucleotide analogue is probably required for this interaction.     

Changes in the activity of alkaline phosphatase and Na K ATPase in the membrane fractions of small-intestinal epithelium in the normal state and in diarrhea

Activity of alkaline phosphatase (apical membrane) and Na+, K+-ATPase (basal membrane) is determined to decrease in the membrane fractions of intestinal epithelium in the diarrheic new born cattle. It is concluded that Na+, K+-ATPase of basal membranes play a fundamental role in Na+ loss by body with diarrhea.     

Characterization of native and reconstituted hydrogen ion pumping adenosinetriphosphatase of chromaffin granules

The ATP-dependent H+ pump from adrenal chromaffin granules is, like the platelet-dense granule H+ pump, essentially insensitive to the mitochondrial ATPase inhibitors sodium azide, efrapeptin, and oligomycin and also insensitive to vanadate and ouabain, agents that inhibit the Na+,K+-ATPase. The chromaffin granule H+ pump is, however, sensitive to low concentrations of NEM (N-ethylmaleimide) and Nbd-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). These transport ATPases may thus belong to a new class of ATP-dependent ion pumps distinct from F1F0-and phosphoenzyme-type ATPases. Comparisons of ATP hydrolysis with ATP-dependent serotonin transport suggest that approximately 80% of the ATPase activity in purified chromaffin granule membranes is coupled to H+ pumping. Most of the remaining ATPase activity is due to contaminating mitochondrial ATPase and Na+,K+-ATPase. When extracted with cholate and octyl glucoside, the H+ pump is solubilized in a monodisperse form that retains NEM-sensitive ATPase activity. When reconstituted into proteoliposomes with crude brain phospholipid, the extracted enzyme recovers ATP-dependent H+ pumping, which shows the same inhibitor sensitivity and nucleotide dependence as the native pump. These data demonstrate that the predominant ATP hydrolase of chromaffin granule membrane is also responsible for ATP-driven amine transport and granule acidification in both native and reconstituted membranes.