The phosphatase activity of the plasma membrane Ca2+ pump. Activation by acidic lipids in the absence of Ca2+ increases the apparent affinity for Mg2+.

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).

Inhibition of rat brain microsomal (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase by periodic acid.

The effects of mild periodate exposure on the kinetics of (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase were studied using rat cerebral microsome preparations. Fifty percent inhibition of both enzyme activities was attained near 3 microM periodate concentrations. This inhibition was biphasic with time. Mg2+-ATPase and Mg2+-p-nitrophenylphosphatase activities were much less inhibited by periodate. Periodate inhibition was partially reversed by dimercaprol and dithiothreitol but not by diffusion. The possible reaction products formic acid, formaldehyde, glyceraldehyde, and acetaldehyde had no inhibitory effects in similar concentrations. Periodate exposure produced no detectable changes in the activation of (Na+ + K+)-ATPase by Na+, K+, Mg2+, or ATP. Residues shared by both (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase are both critical to hydrolytic function and sensitive to mild oxidation by periodate.

Monoclonal antibody to phosphatidylserine inhibits Na+/K(+)-ATPase activity.

A monoclonal IgG, directed to phosphatidylserine (PS1G3), partially (40-50%) inhibited Na+/K(+)-ATPase activity (forward running reaction cycle) without affecting the K0.5 values for Na+,K+ and MgATP. The Hill or interaction coefficients (nH) for Na+ and K+ for this reaction were reduced from 3.0 to 1.6 and from 1.6 to 0.8, respectively. The K(+)-stimulated p-nitrophenylphosphatase activity (p-NPPase), which is a partial reaction sequence of the Na+/K(+)-ATPase system (but in the backward running mode), was inhibited more strongly (about 70%) due to an increase in K+/substrate antagonism. In this system K0.5 and nH values for both p-nitrophenyl phosphate (p-NPP) and K+ were increased by the mAb. At the maximally inhibitory concentration of PS1G3 the Vmax of the p-NPPase was also reduced. Partial reactions, which were inhibited by PS1G3, are: (1) the Na(+)-activated phosphorylation (non-competitive vs. Na+), (2) the Rb+ occlusion (competitive vs. Rb+). Partial reactions not harmed by PS1G3 are: (3) the K(+)-dependent dephosphorylation, (4) the K(+)-dependent E1 + K+<-->E2K transition. We conclude that PtdSer is involved in cation occlusion, possibly by forming part of the access gate.

Calcium inhibition of the ATPase and phosphatase activities of (Na+ + K+)-ATPase.

In experiments performed at 37 degrees C, Ca2+ reversibly inhibits the Na+-and (Na+ + K+)-ATPase activities and the K+-dependent phosphatase activity of (Na+ + K+)-ATPase. With 3 mM ATP, the Na+-ATPase was less sensitive to CaCl2 than the (Na+ + K+)-ATPase activity. With 0.02 mM ATP, the Na+-ATPase and the (Na+ + K+)-ATPase activities were similarly inhibited by CaCl2. The K0.5 for Ca2+ as (Na+ + K+)-ATPase inhibitor depended on the total MgCl2 and ATP concentrations. This Ca2+ inhibition could be a consequence of Ca2+-Mg2+ competition, Ca . ATP-Mg . ATP competition or a combination of both mechanisms. In the presence of Na+ and Mg2+, Ca2+ inhibited the K+-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K+ and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca2+ alone sustained no more than 2% of the (Na+ + K+)-ATPase activity and about 23% of the Na+-ATPase activity observed with Mg2+ and no Ca2+. With 0.003 mM ATP, Ca2+ was able to maintain about 40% of the (Na+ + K+)-ATPase activity and 27% of the Na+-ATPase activity seen in the presence of Mg2+ alone. However, the E2(K)-E1K conformational change did not seem to be affected. Ca2+ inhibition of the K+-dependent rho-nitrophenylphosphatase activity of the (Na+ + K+)-ATPase followed competition kinetics between Ca2+ and Mg2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K+-dependent rho-nitrophenylphosphatase activity as a function of Ca2+ concentration was the same with and without ATP, suggesting that Ca2+ indeed plays the important role in this process. In the absence of Mg2+, Ca2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with rho-nitrophenylphosphate or with acetyl phosphate as substrate.

Effects of polyamines on partial reactions of membrane (Na+ + K+)-ATPase.

Spermine and spermidine inhibit the (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) reaction so that the effect increases as the ionic content due to Na+ and K+ in the reaction is reduced. Several other amines inhibit (Na+ + K+)-ATPase to varying degress and methylglyoxal-bis-(guanylhydrazone) was the most potent inhibitor among those tested. The inhibition by polyamines of the ATPase is uncompetitive with respect to Mg2+ and ATP activation of the reaction. Various naturally occurring polyamines and other amines inhibited Na+ activation of (Na+ + K+)-ATPase as well as Na+-dependent phosphoenzyme formation in an apparently competitive manner with respect to Na+. Likewise, K+-activation of (Na+ + K+)-ATPase as well as K+-p-nitrophenyl phosphatase was inhibited in an apparently competitive manner with respect to K+. Both the cation charge and structure (e.g., aliphatic chain length) may contribute to the inhibitory effects of the amines; however, Na+ sites appear to be more sensitive to cation charge than the aliphatic chain length of the amine, whereas the opposite appears to be true for K+ sites. The results do not indicate a specific effect of polyamines on (Na+ + K+)-ATPase or its partial reactions.

Inhibition of the phosphatase activity of the red cell membrane Ca2+ pump by acidic phospholipids.

The effect of phospholipids was tested on the p-nitrophenylphosphatase activity of the Ca2+ pump. Acidic phospholipids like phosphatidylserine and phosphatidylinositol inhibited the phosphatase activity, while neutral phospholipids like phosphatidylcholine did not. This result contrasts sharply with the known activating effect of acidic phospholipids on the Ca2(+)-ATPase activity of the pump. It is known that the phosphatase activity of the Ca2+ pump can be elicited either by calmodulin and Ca2+ or by ATP and Ca2+. Unlike calmodulin, acidic phospholipids failed to stimulate the phosphatase activity. Furthermore, calmodulin-activated phosphatase was completely inhibited by acidic phospholipids. Maximal inhibition of the ATP-activated phosphatase was only 70%. Inhibition by acidic phospholipids was non-competitive regarding to calmodulin, suggesting that acidic phospholipids and calmodulin do not bind to the same domain of the pump. The presence of Ca2+ was essential for the inhibition, and the apparent affinity for Ca2+ for this effect was increased by acidic phospholipids. Results are consistent with the idea that acidic phospholipids stabilize an enzyme-Ca complex lacking phosphatase activity.

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.

The calmodulin-binding domain as an endogenous inhibitor of the p-nitrophenylphosphatase activity of the Ca2+ pump from human red cells.

Digestion of red cell membranes with chymotrypsin elicited p-nitrophenylphosphatase activity. During digestion, the p-nitrophenylphosphatase appeared in parallel with the activation of the Ca(2+)-ATPase (in the absence of calmodulin). The chymotrypsin-activated p-nitrophenylphosphatase was inhibited by C20W, a 20 amino acid peptide modelled after the sequence of the calmodulin-binding site of the red cell Ca2+ pump (Vorherr et al. (1990) Biochemistry 29, 355-365). On the contrary, the (ATP + Ca(2+)-dependent p-nitrophenylphosphatase activity of intact red cell membranes was not affected by C20W. Ca2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase (Ki for Ca2+ = 2 microM). In the absence of ATP, C20W and Ca2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an 'autoinhibitory domain' of the Ca2+ pump, and that removal of this domain by proteolysis, or its modification by calmodulin binding is the reason for the activation of both the ATPase and the p-nitrophenylphosphatase activity of the pump. The results presented in this paper give new information about the mechanism of the two kinds of p-nitrophenylphosphatase and about the nature of the apparent competition between C20W and Ca2+.

Purification and characterization of digitalislike factors from human plasma.

Increased levels of a humoral inhibitor of active sodium transport have been associated with the response to acute and chronic hypervolemia and various forms of experimental as well as human essential hypertension. In this report, we describe the purification of inhibitors of Na+, K+-adenosine triphosphatase (ATPase) from the plasma of volume-expanded individuals. Of the two amphipathic materials obtained, only one of the factors when present in high concentrations showed the slow time-dependent component of inactivation similar to that of the cardiac glycosides. Inhibition was reduced in the presence of plasma proteins and was freely reversible. Both factors inhibited potassium-dependent p-nitrophenylphosphatase activity and specific [3H]ouabain binding in a manner similar to the cardiac glycosides. In contrast to ouabain and vanadate, however, high concentrations of potassium or norepinephrine, respectively, did not affect the magnitude or kinetic characteristics of inhibition. Structural analysis by mass spectroscopy showed a mass of 444 for factor 1, whereas factor 2 was conclusively identified as lysophosphatidylcholine-gamma-palmitoyl. These factors probably inhibit Na+, K+-ATPase by a nonspecific mechanism involving reversible perturbation of lipid-enzyme interactions required for normal catalytic activity. The significance of these factors as modulators of sodium transport may be limited to pathological states associated with abnormalities in plasma protein binding or lipid metabolism. They do not appear to be directly related to the humorally mediated disturbance of cellular sodium transport in hypertension.

Membrane biochemistry of the ouabain-resistant potassium transport system.

A 6.5-kilobase fragment of genomic DNA from mutant mouse cells under ouabain selection pressure conferred ouabain resistance when transfected into ouabain-sensitive CV1 green monkey fibroblasts. Ouabain resistance was induced in the presence of 10 microM ouabain. Amiloride (500 microM) completely blocked ouabain-insensitive 86Rb+ uptake into these cells. Plasma membranes from these cells demonstrated little sodium-dependent adenosine triphosphatase (ATPase) activity but had potassium-dependent and ouabain-resistant p-nitrophenylphosphatase activity. Like Na+,K+-ATPase this activity was vanadate- and sodium-inhibitable. Also, like the Na+,K+-ATPase, sodium inhibition of the p-nitrophenylphosphatase was reversed by 10 microM adenosine 5'-triphosphate.

Modulation of gastric H+,K+-transporting ATPase function by sodium.

Gastric H+,K+-ATPase activity is not affected by Na+ at pH 7.0 but is significantly stimulated by Na+ at pH 8.5. For the stimulation at the latter pH, the presence of both Na+ and K+ were essential. Contrary the H+,K+-ATPase, the associated K+-pNPPase was inhibited by Na+ at both pH values. Sodium competes with K+ for the K+-pNPPase reaction. Also, unlike the H+, K+-ATPase activity the ATPase-mediated transport of H+ within the gastric microsomal vesicles was inhibited by Na+. For the latter event only the extravesicular and not the intravesicular Na+ was effective. The data suggest that the K+-pNPPase activity does not represent the phosphatase step of the H+,K+-ATPase reaction. In addition, the observed inhibition of vesicular H+ uptake by Na+ appears to be due to the displacement by Na+ of a cytosolic (extravesicular) H+ site responsible for the vectorial translocation of H+.

New azolidinediones as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties.

Insulin resistance in the liver and peripheral tissues together with a pancreatic cell defect are the common causes of type 2 diabetes. It is now appreciated that insulin resistance can result from a defect in the insulin receptor signaling system, at a site post binding of insulin to its receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators of the insulin receptor. Inhibiton of PTPases may be an effective method in the treatment of type 2 diabetes. A series of azolidinediones has been prepared as protein tyrosine phosphatase 1B (PTP1B) inhibitors. Several compounds were potent inhibitors against the recombinant rat and human PTP1B enzymes with submicromolar IC(50) values. Elongated spacers between the azolidinedione moiety and the central aromatic portion of the molecule as well as hydrophobic groups at the vicinity of this aromatic region were very important to the inhibitory activity. Oxadiazolidinediones 87 and 88 and the corresponding acetic acid analogues 119 and 120 were the best h-PTP1B inhibitors with IC(50) values in the range of 0.12-0.3 microM. Several compounds normalized plasma glucose and insulin levels in the ob/ob and db/db diabetic mouse models.

Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.

Mechanism of gastric antisecretory effect of SCH 28080.

The mechanism of the gastric antisecretory action of SCH 28080 has been studied utilizing two different in vitro test systems, isolated and enriched parietal cells from the guinea-pig and guinea-pig gastric membranes purified and enriched with K+/H+-ATPase. In guinea-pig isolated and enriched parietal cells SCH 28080 inhibited the acid response to histamine and high K+ concentrations with IC50 values not significantly different from each other. SCH 28080 inhibited the purified K+/H+-ATPase measured in the presence of 5 mM KCl with an IC50 value of 1.3 microM. Kinetic studies indicated a competitive inhibition of ATPase by SCH 28080 with respect to K+. Studies on Na+/K+-ATPase showed that this enzyme was only slightly depressed by SCH 28080. It is concluded that SCH 28080 acts with high selectivity on the parietal cell K%/H+-ATPase, establishing its antisecretory effect by a competitive interaction with the high affinity K+-site of the gastric ATPase.

Inhibition of Na+,K(+)-ATPase by 1,2,3,4,6-penta-O-galloyl-beta-D-glucose, a major constituent of both moutan cortex and Paeoniae radix.

The inhibition of Na+,K(+)-ATPase activity by various constituents of Moutan Cortex and Paeoniae Radix was studied. 1,2,3,4,6-Penta-O-galloyl-beta-D-glucose (PGG), a major component of both crude drugs, strongly inhibited Na+,K(+)-ATPase activity (IC50 = 2.5 x 10(-6) M), whereas galloylpaeoniflorin, benzoic acid, and catechin were weakly inhibitory, and albiflorin, oxypaeoniflorin, paeoniflorin, paconol, and phenol were ineffective. The inhibition of Na+,K(+)-ATPase activity by PGG was decreased in the presence of BSA or phospholipids. The inhibition mode of PGG was noncompetitive with respect to ATP. The K0.5 value for Na+ was increased by the addition of PGG from 9.1 to 12.3 mM, whereas that for K+ was not altered. PGG also inhibited K(+)-dependent p-nitrophenyl phosphatase activity with an IC50 value of 5.3 x 10(-6) M, and the extent of the inhibition increased at higher concentrations of K+. The K0.5 value for K+ was decreased by the addition of PGG from 3.3 to 2.0 mM. These results suggested that the inhibition of Na+,K(+)-ATPase activity is caused by interaction of PGG with the enzyme in the E2 state. The inhibitory effect of Moutan Cortex or Paeoniae Radix is considered to be mainly attributable to PGG.

Interaction of beta-eudesmol with Na+,K(+)-ATPase: inhibition of K(+)-pNPPase activity.

The effect of beta-eudesmol, one of the major components in So-jutsu (Atractylodis Lanceae Rhizoma), on K(+)-dependent p-nitrophenyl phosphatase (K(+)-pNPPase) activity was studied. It inhibited K(+)-pNPPase activity with an I50 value of 4.1 x 10(-4) M. The inhibition rate decreased as the K+ concentration was increased, whereas greater inhibition was observed with high concentrations of either Na+ or ATP. The Ki values for Na+ in the presence of 0, 0.1 and 1 mM ATP were 140, 260 and 310 mM, respectively, but with the addition of beta-eudesmol, these values decreased to 90 mM regardless of the ATP concentration. This study on K(+)-pNPPase activity supports the conclusion obtained from the study on Na+,K(+)-ATPase activity (Satoh K et al., Biochem Pharmacol 44: 373-378, 1992) that is, beta-eudesmol interacts with enzyme in the Na.E1 form and inhibits the reaction step Na.E1----Na.E1-P. Furthermore, in the study of the effects of K+ and beta-eudesmol on K(+)-pNPPase activity, it was confirmed that beta-eudesmol prevents the conformational change of Na.E1----K.E2.

Inhibition of dog kidney Na+, K+-ATPase activity by procaine, tetracaine and dibucaine.

The potency of local anesthetics as inhibitors of Na+, K+-ATPase and K+- NPPase activities correlated with lipid solubility. The order of potencies was: dibucaine greater than tetracaine much greater than procaine. Na+-ATPase activity was remarkably more sensitive to inhibition by tetracaine and procaine, and inhibitory potency did not correlate with lipid solubility. The order of potencies for inhibition of Na+-ATPase activity was: tetracaine greater than dibucaine greater than procaine. We examined interactions between the local anesthetics and monovalent cations in an attempt to explain this observation. Inhibition of Na+-K+-ATPase by tetracaine and dibucaine was competitive with respect to Na+, and inhibition of Na+-ATPase activity by all three agents was competitive with respect to Na+. Inhibition of Na+, K+-ATPase activity by procaine and tetracaine was competitive with respect to K+, and inhibition of K+- NPPase activity by all three agents was competitive with respect to K+. Dibucaine, the most lipid soluble agent, was equipotent as an inhibitor of all three activities and was generally less effective as a competitor with respect to activation by monovalent cations. These results suggest that dibucaine may interact nonspecifically with membrane lipids to inhibit enzyme activity whereas less lipid soluble agents, such as tetracaine and procaine, may interact more selectively with cation binding sites. It appears that the presence of K+ in the assay medium specifically decreases the inhibitory potency of tetracaine and procaine. Direct competition between these agents and K+ may prevent inhibition or, alternately, the presence of K+ may convert the enzyme to a conformation less susceptible to inhibition by agents of low to intermediate lipid solubility.

Specific inhibition of Na+,K(+)-ATPase activity by atractylon, a major component of byaku-jutsu, by interaction with enzyme in the E2 state.

Atractylon, a major component of the crude drug "Byaku-jutsu" (rhizomes of Atractylodes japonica), strongly inhibited Na+,K(+)-ATPase activity with an I50 value of 8.9 x 10(-6) M. It also inhibited Mg(2+)-ATPase, H+,K(+)-ATPase, H(+)-ATPase and Ca(2+)-ATPase activities, but less potently. No effects on alkaline and acid phosphatase activities were observed. The inhibition of Na+,K(+)-ATPase activity by atractylon was noncompetitive with respect to ATP and was greater with increasing K+ concentration, whereas it was not affected by Na+ concentration. The activity of K(+)-dependent p-nitrophenyl phosphatase, a partial reaction of Na+,K(+)-ATPase, was inhibited noncompetitively with respect to substrate (I50 value of 1.8 x 10(-5) M), and the inhibition rate was independent of the K+ concentration. Furthermore, atractylon increased the Ki value for Na+ from 130 to 190 mM, but did not alter the Ki value for ATP. Inhibition of the phosphoenzyme formation by atractylon was greater at 0.1 M than at 1 M NaCl. K(+)-dependent dephosphorylation (E2-P to K.E2) was inhibited by atractylon, whereas ADP-sensitive (Na.E1-P to Na.E1) and non-specific dephosphorylation steps were not affected. These results suggest that atractylon, a specific inhibitor of Na+,K(+)-ATPase, interacts with enzyme in the E2 state and inhibits the reaction step from E2-P to K.E2.

SCH 28080 is a lumenally acting, K+-site inhibitor of the gastric (H+ + K+)-ATPase.

SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo[1,2-a] pyridine-3-acetonitrile) is an effective inhibitor of acid secretion in vivo and is a reversible, K+-competitive inhibitor of the gastric (H+ + K+)-ATPase in vitro. The actions of SCH 28080 have been studied on gastric vesicle preparations containing the (H+ + K+)-ATPase. At pH 7, inhibition was competitive with respect to K+ for both ATPase (Ki = 24 nM) and pNPPase (Ki = 275 nM) activities. A close analogue of SCH 28080 (methylated in the 1-N position), that was not expected to cross membranes freely, inhibited ATPase and pNPPase activity less effectively in intact vesicle preparations, where the lumenal (extracellular) face of the membrane was not directly accessible. This suggested that SCH 28080 inhibited both enzyme activities at a lumenal site on the enzyme. Being a protonatable weak base (pKa = 5.6), SCH 28080 would be expected to accumulate on the lumenal, acidic side of the parietal cell membrane in its protonated form. The potency of SCH 28080, relative to that of the "non-protonatable" analogue, increased at low pH, commensurate with the proportion of SCH 28080 in the protonated form. Thus the accumulating protonated form was the active inhibitory species. SCH 28080 (50 nM) blocked the rapid, K+-stimulated dephosphorylation of the catalytic phosphoenzyme intermediate of the (H+ + K+)-ATPase at room temperature. At 4 degrees, higher concentrations of the inhibitor were required, suggesting that the rate of inhibitor binding was slow at low temperatures.