Role of cell surface mannose residues in host cell invasion by Trypanosoma cruzi

The cholesterol content of human erythrocyte membranes has been modified by incubation of intact cells with sonicated egg phosphatidylcholine/cholesterol vesicles and with egg phosphatidylcholine vesicles. (Na+ + K+)-ATPase ATP hydrolyzing activity was measured as a function of membrane cholesterol content. High membrane cholesterol inhibits the ATPase activity of the enzyme and low membrane cholesterol activates that enzyme activity. The most likely mechanism of inhibition is suggested to comprise direct cholesterol-protein interactions which lead to a low activity conformation. Ouabain binding studies show that the inhibition is not due to a loss of enzyme from the membrane.     

Uptake of aminoglycoside antibiotics into brush-border membrane vesicles and inhibition of (Na+ + K+)-ATPase activity of basolateral membrane

The effects of aminoglycoside antibiotics on plasma membranes were studied using rat renal basolateral and brush-border membrane vesicles. 3',4'-Dideoxykanamycin was bound to the basolateral membrane and brush-border membrane vesicles. They had a single class of binding sites with nearly the same constant, and the basolateral membrane vesicles had more binding sites than those of the brush-border membrane. Dideoxykanamycin B was transported into the intravesicular space of brush-border membrane vesicles, but not into that of basolateral membrane vesicles. The (Na+ + K+)-ATPase activity of the plasma membrane fraction prepared from the kidney of rat administered with dideoxykanamycin B intravenously decreased significantly. Aminoglycoside antibiotics entrapped in the basolateral membrane vesicles inhibited (Na+ + K+)-ATPase activity, but those added to the basolateral membrane vesicles externally failed to do so. The activity of (Na+ + K+)-ATPase was non-competitively inhibited by gentamicin. It is thus concluded that aminoglycoside antibiotics are taken up into the renal proximal tubular cells across the brush-border membrane and inhibit the (Na+ + K+)-ATPase activity of basolateral membrane. This inhibition may possibly disrupt the balance of cellular electrolytes, leading to a cellular dysfunction, and consequently to the development of aminoglycoside antibiotics' nephrotoxicity.     

Na+-stimulated ATPase activities in basolateral plasma membranes from guinea-pig small intestinal epithelial cells

Two ATPase activities, a Na+-ATPase and a (Na+ + K+)-ATPase, have been found associated with sheets of basolateral plasma membranes from guinea-pig small intestinal epithelial cells. The specific activity of the former is 10-15% of the latter. The two ATPase activities differ in their affinity for Na+, their optimal pH, their K+ requirement and particularly in their behaviour in the presence of some inhibitors and of Ca2+. Thus the Na+-ATPase is refractory to ouabain but it is strongly inhibited by ethacrynic acid and furosemide, whilst the (Na+ + K+)-ATPase is totally suppressed by ouabain, partially by ethacrynic acid and refractory to furosemide. In addition, the Na+-ATPase is activated by micromolar concentrations of calcium and by resuspension of the membrane preparation at pH 7.8. The Na+-ATPase is only stimulated by sodium and to a lesser extent by lithium; however, this stimulation is independent of the anion accompanying Na+. The latter rules out the participation of an anionic ATPase. The relation between the characteristics of the sodium transport mechanism in basolateral membrane vesicles (Del Castillo, J.R. and Robinson, J.W.L. (1983) Experientia 39,631) and those of the two ATPase activities present in the same membranes, allow us to postulate the existence of two separate sodium pumps in this membranes. Each pump would derive the necessary energy for active ion transport from the hydrolysis of ATP, catalyzed by different ATPase systems.     

Isolation of basolateral and brush-border membranes from the rabbit kidney cortex. Vesicle integrity and membrane sidedness of the basolateral fraction

A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na+ + K+)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35-50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na+ + K+)-ATPase activity and [3H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na+ + K+)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na+ and K+. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation.     

Oxidative stress reduces transintestinal transports and (Na+, K+) -ATPase activity in rat jejunum

Because oxidative stress is a component of gastrointestinal injury, we investigated the effect of H(2)O(2) on transintestinal transport using isolated rat jejunum incubated in vitro. Millimolar concentrations of H(2)O(2) inhibited all the tested parameters without inducing any cytotoxic effect. Electrophysiological experiments indicated that H(2)O(2) decreases significantly both short circuit current and transepithelial electrical potential difference without affecting transepithelial resistance. The possibility that H(2)O(2) could influence (Na+, K+) -ATPase activity was explored using isolated basolateral membranes. Besides H(2)O(2), free radicals (O(2)(*-), HO*) were generated using different iron-dependent and independent systems; (Na+, K+) -ATPase activity was inhibited after membrane exposure to all ROS tested. The inhibition was prevented by allopurinol, superoxide dismutase or desferrioxamine. Western blot analysis showed a decreased expression of the alpha(1)-subunit of (Na+, K+) -ATPase. We conclude that H(2)O(2) may be a modulator of jejunal ion and water transport by multiple mechanisms, among which a significant inhibition of the basolateral (Na+, K+) -ATPase.     

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.     

Transverse tubule Mg(2+)-ATPase of skeletal muscle. Evidence for extracellular orientation of the chicken and rabbit enzymes.

The orientation of the enzyme Mg(2+)-ATPase (EC 3.6.1.3) in the transverse tubule (TT) membranes of skeletal muscle was investigated using highly purified chicken and rabbit TT vesicles. The percentage of sealed vesicles present in these preparations averaged 88 and 78%, respectively, as calculated from the detergent-induced increase in ouabain-sensitive (Na+, K+)-ATPase activity, ATP-dependent ouabain binding, and lactate dehydrogenase activity (sarcoplasmic enzyme trapped in the TT vesicles). Sidedness of the sealed vesicles, estimated from latency of 5'-nucleotidase, acetylcholinesterase, and adenylate cyclase, was predominantly right-side out (69-76%, chicken TT and 62-70%, rabbit TT). In both chicken and rabbit native vesicles, high Mg(2+)-ATPase activity was detected by addition of ATP to the extravesicular medium; this activity was increased 14-12% by alamethicin pointing to the external localization of the active site. Furthermore, the enzymatic activity resulted partially inhibited by treatment of the chicken TT vesicles with proteinase K or p-hydroxymercuribenzoate. Concanavalin A stimulated 4-fold the chicken TT Mg(2+)-ATPase activity, an effect not potentiated by detergent permeabilization of the intact vesicles, indicating that lectin-binding sites were also solvent accessible. This stimulatory effect was not observed in native or permeabilized rabbit TT vesicles. From these results we conclude that the TT Mg(2+)-ATPase is an ectoenzyme with its nucleotide-hydrolyzing site and glycosylated regions facing the extracellular space. Inhibitors of ion-motive ATPases did not modify the enzyme activity, suggesting a different physiological role for the TT Mg(2+)-ATPase which may be involved in the regulation of muscle fiber functions affected by extracellular ATP levels.     

Sodium transport in basolateral membrane vesicles from rat enterocytes

Basolateral membranes purified from rat jejunal enterocytes and enriched 14 times in (Na, K)-ATPase, are present as unsealed and right side out (RSO) or inside out (IO) vesicles in the ratio 2:2:1, as determined by detergent activation of ATPase activity. Entrance of 1 mM Na into basolateral membrane vesicles was measured in the presence and in the absence of 5 mM ATP by a rapid filtration technique, under different experimental conditions. Carrier-mediated Na transport across the basolateral membrane can be trans-stimulated and cis-inhibited by K and further stimulated by ATP (activation of the Na pump). The ATP effect can be suppressed by vanadate and strophanthidin and enhanced by bleomycin (19% increase), which positively also acts on (Na, K)-ATPase activity (16% increase). In addition to the Na pump this study demonstrates the existence of a carrier-mediated Na transport trans-stimulated by K. There appears to be no cotransport of Na-K.     

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.     

A quantitative immunocytochemical study of Na+,K+-ATPase in rat hepatocytes after STZ-induced diabetes and dietary fish oil supplementation.

Because diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na+,K+-ATPase. In this study we documented the effects of streptozotocin (STZ)-induced diabetes on hepatic Na+,K+-ATPase catalytic alpha1-subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic alpha1-subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na+, K+-ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the alpha1-subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na+,K+-ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.     

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.     

A monoclonal antibody against a native conformation of the porcine renal Na+/K(+)-ATPase alpha-subunit protein

A monoclonal antibody (mAb50c) against the native porcine renal Na+/K(+)-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K(+)-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na+/K(+)-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na+, ATP or Mg2+ (E1 form), slightly less in the presence of K+ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na+, Mg2+ and ATP. The antibody inhibited both the Na+,K(+)-ATPase activity and the K(+)-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na(+)-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the alpha-subunit.     

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.     

Inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells

A method for preparation of highly purified basolateral plasma membranes from rat kidney proximal tubular cells is reported. These membranes were assayed for the presence of vesicles as well as for their orientation. (Na+ + K+)-ATPase activity and [3H]ouabain binding studies with membranes treated with or without SDS revealed that the preparation consisted of almost 100% vesicles. The percentage of inside-out vesicles was found to be approx. 70%. This percentage was determined measuring the (Na+ + K+)-ATPase activity in K+-loaded vesicles and in membranes treated with or without trypsin and SDS. These membranes represent a very efficient tool to assay the correlation between active transport and ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells.     

Effect of a NaCl gradient on the transport of para-aminohippuric acid into the vesicles of the basolateral membrane of the kidney cortex

Basolateral membrane vesicles were isolated from the rat kidney cortex by a modified method of cation precipitation. Different steps of preparation were analysed using the marker enzymes: Na+,K+-ATPase (for basolateral membrane), alkaline phosphatase (for apical membrane), glucose-6-phosphatase (for membranes of endoplasmic reticulum) and succinate dehydrogenase (for mitochondria). The basolateral membrane was purified by a 8-9-fold treatment with Na+,K+-ATPase, while other membrane contaminations were as low as 2% (as compared to homogenate). The transport of 3H-p-aminohippurate (3H-PAH) by basolateral membrane vesicles was measured under different experimental conditions. The 3H-PAH uptake was found to be Na-gradient dependent. The initial rate of 3H-PAH uptake in the presence of NaCl gradient (500 pM/mg X min) was higher than without the gradient (88 pM/mg X min). It is concluded that the PAH transfer across the basolateral membrane may be energized by the Na+ chemical gradient.     

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.     

Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles

A mechanism for the activating effect of alamethicin on membrane enzymes was investigated, using a purified preparation of cardiac sarcolemmal vesicles. (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities were measured. alamethicin increased ouabain-sensitive (Na+,K+)-ATPase activity of sarcolemmal vesicles 5- to 7-fold and adenylate cyclase activity 2.5- to 4-fold. Adenylate cyclase retained its sensitivity to the beta-adrenergic agonist isoproterenol after membranes were treated with alamethicin. Alamethicin caused a 4- to 6-fold increase in the number of detectable (Na+,K+)-ATPase enzymic sites, but no increase ws noted for the number of muscarinic-cholinergic receptor-binding sites. Phosphorylation of endogenous proteins of sarcolemmal vesicles by an intrinsic cAMP-dependent protein kinase activity was stimulated 5- to 7-fold by alamethicin. The regulatory subunit of the membrane-bound cAMP-dependent protein kinase was labeled with the photoaffinity probe 8-azido-adenosine 3':5'[32P]monophosphate (8-N3-[32P]cAMP), and it migrated with an apparent molecular weight of 55,000 in sodium dodecyl sulfate polyacrylamide gels. Alamethicin stimulated autophosphorylation of the regulatory subunit by [gamma-32P]ATP 6-fold and incorporation of of 8-N3-[32P]cAMP into the subunit 2.6-fold. The results suggest that alamethicin disrupts membrane barriers of sarcolemmal vesicles, which are mostly right side out, giving substrates and activators access to enzymic sites in the interior of the vesicles, while preserving functional coupling of enzymes to their effectors.     

Prolactin, an activator of epithelial Na+ channel, inhibits basolateral K+ channels in adult tree frog skin

Adult amphibian skin actively transports Na+ from its apical to basolateral side while in turn, K+ is recycled through Na+, K+-ATPase and K+ channels located in the basolateral membrane. We previously found that PRL stimulates Na+ transport in the skin of the adult tree frog (Hyla arborea japonica) via an increase in the open-channel density of the epithelial Na+ channel (ENaC). If PRL also activates basolateral K+ channels, this activation would help to stimulate Na+ transport, too. Whether PRL does indeed stimulate basolateral K+ channels in the adult tree frog was examined by measuring the short-circuit current across nystatin-treated skin. Both tolbutamide, a K(ATP) channel blocker, and tetrapentylammonium (TPA), a KCa channel blocker, blocked the current, the effect of TPA being more powerful than that of tolbutamide. Contrary to expectation, PRL inhibited the basolateral K+ channels in this skin. In the presence of basolateral amiloride, PRL still inhibited the basolateral K+ current, suggesting that the (Na+)-H+ exchanger located in the basolateral membrane does not mediate the inhibitory effect of PRL on the basolateral K+ channels in Hyla.     

Topological localization of proteolytic sites of sodium and potassium ion stimulated adenosinetriphosphatase

The (Na+ and K+)-stimulated adenosinetriphosphatase [(Na+,K+)-ATPase] consists of two different polypeptides, alpha and beta, both of which are embedded in the plasma membrane. The alpha chain from dog kidney (Na+,K+)-ATPase can be hydrolyzed at specific sites by trypsin and chymotrypsin [Castro, J., & Farley, R. A. (1979) J. Biol. Chem. 254, 2221-2228]. In order to position these sites with respect to the lipid bilayer, we have treated sealed, inside out vesicles from human red cells and unsealed kidney enzyme membranes with trypsin and chymotrypsin and have used ouabain-stimulated phosphorylation to identify the (Na+,K+)-ATPase and its fragments. All of the proteolytic sites observed in the kidney membranes are accessible in the inside out vesicles. The ouabain-inhibitable uptake of 86Rb+ in human red blood cells is resistant to externally added chymotrypsin. These results indicate that the proteolytic sites of the (Na+,K+)-ATPase are exposed on the cytoplasmic side of the membrane.     

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.