Modification of gastric (H+ + K+)-ATPase with pyridoxal 5'-phosphate

Pig gastric membrane vesicles enriched in (H+ + K+)-ATPase were covalently modified with pyridoxal 5'-phosphate (PLP). The modification resulted in inhibition of K+-dependent ATP hydrolysis, formation of phosphoenzyme and ATP-driven H+-uptake catalyzed by (H+ + K+)-ATPase. ATP, ADP, and adenyl-5'-yl imidodiphosphate were protective ligands, whereas Mg2+ and K+ were not. Specific PLP-binding of about 4.5 nmol/mg membrane protein was necessary for complete inhibition of the enzyme activity, indicating that the stoichiometry of PLP-binding to the enzyme was about 1:1. Limited proteolysis of the enzyme modified with [3H]PLP by trypsin suggests that PLP specifically modifies the lysine residue located in the 16-kDa fragment of the enzyme cleaved by trypsin. These results suggested that PLP binds to a specific lysine residue in the nucleotide-binding site or a region in its vicinity and inhibits the substrate binding or phosphorylation step of (H+ + K+)-ATPase.     

Redistribution and characterization of (H+ + K+)-ATPase membranes from resting and stimulated gastric parietal cells.

When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+)-ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation.     

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.     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

Studies on (K+ + H+)-ATPase. I. Essential arginine residue in its substrate binding center

1. A membrane vesicle fraction containing a high (K+ + H+)-ATPase activity was isolated from porcine gastric mucosa. The enzyme has a pH optimum of 7.0 and is stimulated by T1+, K+, Rb+ and NH4+ with KA values of 0.13, 2.7, 7.6 and 26 mM, respectively, at this pH. 2. Incubation of the isolated membrane fraction with butanedione leads to inactivation of the (K+ + H+)-ATPase activity. The pH-dependence of the (K+ + H+)-ATPase activity. The pH-dependence of the inactivation and the reversibility of the reaction, observed after removal of excess butanedione and borate, indicate that modification of arginine is involved. 3. The inactivation of (K+ + H+)-ATPase activity by butanedione is time-dependent and follows second-order kinetics. From the dependence of the inactivation rate on the reagent concentration it appears that a single arginine residue is involved in the inactivation of the (K+ + H+)-ATPase activity. 4. ATP, deoxy-ATP, ADP and adenylyl imidodiphosphate (AMPPNP), but not CTP, GTP and ITP which are poor substrates, protect the enzyme against butanedione inactivation, suggesting that the essential arginine residue is located in the ATP binding centre. 5. In the presence of Mg2+ the butanedione inactivation is increased, and the protection by ATP, deoxy-ATP and ADP (but not that by AMPPNP) is less pronounced. This suggests that Mg2+ induces a conformational change in the enzyme, exposing the arginine group and coinciding with phosphorylation and subsequent release of ADP from its binding site.     

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.     

Effect of lysophosphatidylcholine on K+ transport in rat heavy gastric membranes enriched with (H+-K+)-ATPase

K+- and ATP-dependent H+-accumulation in rat heavy gastric membrane vesicles enriched with (H+-K+)-ATPase was markedly stimulated by amphiphiles like lysophosphatidylcholine and Zwittergent 3-14 at concentrations of 10(-5) M. Their stimulatory effect was dependent on K+-concentration in the medium and was abolished by SCH 28,080, a specific inhibitor of (H+-K+)-ATPase. Lysophosphatidylcholine at the optimal dose (3 X 10(-5) M) showed dual effects on K+-dependent membrane functions; it stimulated the rate of K+-uptake by nearly 60%, but partially inhibited SCH 28,080-sensitive and K+-dependent ATP-hydrolysis (about 20% reduction). These data indicate that H+-pumping through (H+-K+)-ATPase in the inside-out gastric membrane vesicles was facilitated by the stimulatory effect of lysophosphatidylcholine on membrane K+-transport in spite of its partial inhibition of ATP-hydrolysis. It appears that the rate limiting step for operation of the ATPase is the availability of K+ ions in the luminal side of the pump. We propose that ionic amphiphiles may modulate K+-transport in rat heavy gastric membranes through specific interactions with the putative K+-transporter.     

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.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

Transport ratios of reconstituted (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol vesicles by means of a freeze-thaw-sonication procedure. The passive and active transport mediated by these vesicles were measured (Skrabanja, A.T.P., Asty, P., Soumarmon, A., De Pont, J.J.H.H.M. and Lewin, M.J.M. (1986) Biochim. Biophys. Acta 860, 131-136). To determine real initial velocities, the proteoliposomes were separated from non-incorporated enzyme, by means of centrifugation on a sucrose gradient. The purified proteoliposomes were used to measure active H+ and Rb+ transport, giving at room-temperature velocities of 46.3 and 42.5 mumol per mg per h, respectively. A transport ratio of two cations per ATP hydrolyzed was also measured. These figures indicate that the enzyme catalyzes an electroneutral H+-Rb+ exchange.     

Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase

A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [14C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [14C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump.     

K+-stimulated p-nitrophenyl phosphatase is not a partial reaction of the gastric (H+ + K+)-transporting ATPase. Evidence supporting a new model for the univalent-cation-transporting ATPase systems.

Studies with intact and lysed gastric microsomal vesicles demonstrate that there are two pNPP (p-nitrophenyl phosphate)-and one ATP-hydrolytic sites within the gastric H+, K+-ATPase [(H+ + K+)-transporting ATPase] complex. Whereas the ATPase site is located exclusively on the vesicle exterior, the pNPPase sites are distributed equally on both sides of the bilayer. Competition by ATP for the pNPPase reaction on the vesicle exterior suggests that both ATP and pNPP are hydrolysed at the same catalytic site present at the outside surface of the intact vesicles. However, a biphasic inhibition of the K+-pNPPase (K+-stimulated pNPPase) by ATP in the lysed vesicles suggest the pNPPase site of the vesicle interior to have very low affinity (Ki approximately equal to 1.2 mM) for ATP compared with the vesicle exterior (Ki approximately equal to 0.2 mM). Studies with spermine, which competes with K+ for the K+-pNPPase reaction without inhibiting the H+, K+-ATPase, suggest there are two separate K+ sites for the pNPPase reaction and another distinct K+ site for the ATPase reaction. In contrast with the K+ site for the ATPase, which is located opposite to the catalytic site across the bilayer, both the K+ and the catalytic site for the pNPPase are located on the same side. The data clearly demonstrate that the pNPPase is not a manifestation of the phosphatase step of the total H+, K+-ATPase reaction. The K+-pNPPase associated with the Na+, K+-ATPase also has properties strikingly similar to the gastric K+-pNPPase system, suggesting a resemblance in the basic operating principle of the two ion-transporting enzymes. A unified model has been proposed to explain the present data and many other observations reported in the literature for the ATPase-mediated transport of univalent cations.     

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.     

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).     

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.     

Direct evidence for the cytoplasmic location of the NH2- and COOH-terminal ends of the Neurospora crassa plasma membrane H+-ATPase

Reconstituted proteoliposomes containing Neurospora plasma membrane H+-ATPase molecules oriented predominantly with their cytoplasmic portion facing outward have been used to determine the location of the NH2 and COOH termini of the H+-ATPase relative to the lipid bilayer. Treatment of the proteoliposomes with trypsin in the presence of the H+-ATPase ligands Mg2+, ATP, and vanadate produces approximately 97-, 95-, and 88-kDa truncated forms of the H+-ATPase similar to those already known to result from cleavage at Lys24, Lys36, and Arg73 at the NH2-terminal end of the molecule. These results establish that the NH2-terminal end of the H+-ATPase polypeptide chain is located on the cytoplasmic side of the membrane. Treatment of the same proteoliposome preparation with trypsin in the absence of ligands releases approximately 50 water-soluble peptides from the proteoliposomes. Separation of the released peptides by high performance liquid chromatography and spectral analysis of the purified peptides identified only a few peptides with the properties expected of a COOH-terminal, tryptic undecapeptide with the sequence SLEDFVVSLQR, and NH2-terminal amino acid sequence analysis identified this peptide among the possible candidates. Quantitative considerations indicate that this peptide must have come from H+-ATPase molecules oriented with their cytoplasmic portion facing outward, and could not have originated from a minor population of H+-ATPase molecules of reverse orientation. These results directly establish that the COOH-terminal end of the H+-ATPase is also located on the cytoplasmic side of the membrane. These findings are important for elucidating the topography of the membrane-bound H+-ATPase and are possibly relevant to the topography of other aspartyl-phosphoryl-enzyme intermediate ATPases as well.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

The NH2 terminus of the (Ca2+ + Mg2+)-adenosine triphosphatase is located on the cytoplasmic surface of the sarcoplasmic reticulum membrane

The (Ca2+ + Mg2+)-adenosine triphosphatase (ATPase) of sarcoplasmic reticulum contains a cysteine residue at position 12 of its sequence. This sulfhydryl group was 1 out of a total of 10-11 that were labeled by treatment of sarcoplasmic reticulum vesicles with N-[3H]ethylmaleimide under saturating conditions. This was shown by isolating a 31-residue NH2-terminal peptide from a tryptic digest of the succinylated ATPase, prepared from N-[3H]ethylmaleimide-labeled vesicles. Reaction of the vesicles with glutathione maleimide, parachloromercuribenzoic acid, or parachloromercuriphenyl sulfonic acid, membrane-impermeant reagents, prevented further reaction of sulfhydryl groups with N-ethylmaleimide. This result indicates that all sulfhydryl groups that are reactive with N-ethylmaleimide are on the outside of the vesicles. Since Cys12 is located in a hydrophilic NH2-terminal portion of the ATPase, the labeling results suggest that the NH2 terminus of the ATPase is on the cytoplasmic side of the membrane. These results are consistent with earlier observations (Reithmeier, R. A. F., de Leon, S., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 11839-11846) that the (Ca2+ + Mg2+)-ATPase is synthesized without an NH2-terminal signal sequence.     

Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.

A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174----Phe mutation (defective H(+)-ATPase), but beta Gly150----Ser substitution did not have this effect. A single mutation (beta Gly149----Ser or beta Gly150----Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149----Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149----Ser or beta Gly150----Ser mutation with the beta Ser174----Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156----Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.