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.     

Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

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.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.     

The presence of two (Na+ + K+)-ATPase inhibitors in equine muscle ATP: vanadate nad a dithioerythritol-dependent inhibitor.

A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 micrometer in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate is increased by free Mg2+. The inhibition is half maximally reversed by 250 micrometer epinephrine. Equine muscle ATP was also found to contain a second (Na+ + K+)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 micrometer epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na+ + K+)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na+ + K+)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase.     

The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump

Inhibition by vanadate of the K+-dependent p-nitrophenylphosphatase activity catalyzed by the (Na+ + K+)-ATPase partially purified from pig kidney showed competitive behavior with the substrate, K+ and Mg2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K+-dependent p-nitrophenyl phosphate hydrolysis (Na+, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p-nitrophenyl phosphate and Mg2+, or when the protective ligands were tested alone, the activation of p-nitrophenyl phosphate hydrolysis by K+ followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K+ on the p-nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K+ concentration, the p-nitrophenylphosphatase activity declined at high K+ concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na+ (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K+ - K+ exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na+ plus ATP, K+ promoted vanadate inhibition of p-nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na+ plus ATP, the biphasic response of p-nitrophenyl phosphate hydrolysis is due to K+ acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb+ influx as a function of the external Rb+ concentration failed to develop when there was no Na+ in the extracellular media. In addition, in the absence of extracellular Na+, external Rb+ did not influence the magnitude of inhibition. The present findings indicate that external K+ favors vanadate inhibition by displacing Na+ from unspecified extracellular membrane sites.     

Vanadate sensitivity of Na+, K+-ATPase from Schistosoma mansoni and its modulation by Na+, K+ and Mg2+

Vanadate inhibitory effects on Na+, K+-ATPases from carcass of Schistosoma mansoni and from lamb kidney outer medulla were compared in the presence of various concentrations of Na+, K+ and Mg2+. Depending on the ionic conditions, the schistosomal Na+, K+-ATPase was 2.4- to 175-fold less sensitive to vanadate than the lamb kidney enzyme. In 100 mM Na+, 3 mM K+ and 3 mM Mg2+, schistosomal Na+, K+-ATPase was surprisingly resistant to vanadate (I50 = 944 microM). The difference in vanadate sensitivity between schistosomal and lamb Na+, K+-ATPases may be due to a species difference in the efficacy of Na+, K+ and Mg2+ in promoting conformational changes between E1 and E2 forms of the enzyme.     

Local anesthetics induce fast Ca2+ efflux through a nonenergized state of the sarcoplasmic reticulum Ca(2+)-ATPase.

The effect of the local anesthetics SKF 525-A, dibucaine, tetracaine, procaine, and benzocaine on sarcoplasmic reticulum vesicles was studied. All the anesthetics tested inhibited the phosphorylation of the Ca(2+)-ATPase by Pi in a competitive manner. Tertiary amine and positively charged anesthetics, in addition to competing with Pi, also decreased the apparent affinity of the ATPase for Mg2+. There was a good correlation between the octanol/water partition coefficients and the inhibitory activity of the different anesthetics. All the anesthetics tested induced a 5- to 10-fold increase in the rate of Ca2+ efflux. This was promoted by the same drug concentration that inhibited the phosphorylation of the ATPase by Pi. The effect on Ca2+ efflux was antagonized by the ligands of the ATPase (Mg2+, K+, Ca2+, MgATP, and ADP) and by the organic polyamines ruthenium red, spermine, spermidine, and putrescine. The natural anion heparin was found to potentiate the effect of the positively charged anesthetics on the rate of Ca2+ efflux. It is concluded that the local anesthetics increase the Ca2+ efflux through a nonenergized state of the Ca(2+)-ATPase, rather than promoting a nonspecific Ca2+ leakage through the membrane.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mg2+-ATPase as a membrane ecto-enzyme of human granulocytes. Inhibitors, activators and response to phagocytosis.

(1) The Mg2+-ATPase of purified human granulocytes is located at the plasma membrane. Thus, no additional enzyme activity was detected when the cells were disrupted. Moreover, the Mg2+-ATPase activity of intact cells was inhibited by such poorly permeant reagents as diazotized sulfanilic acid and suramin. Finally, the enzyme activity of cell homogenates was recovered in particulate fractions. (2)The surface Mg2+-ATPase of human granulocytes had an apparent Km of 50 microns for ATP and displayed substrate inhibition. (3) The enzyme was not affected by ouabain, but was inhibited by N-ethyl malemide, sodium meta-periodate, suramin and diazotized sulfanilic acid. The enzyme was activated by cytochalasins B and D and by UDP. Activation by UDP was characterized by changes in the enzyme's apparent Km and V and by belief of substrate inhibition. (4)Internalization of surface membranes subsequent to phagocytosis of suitable particles did not result in depletion of Mg2+-ATPase from the cell surface. The enzyme activity did not decrease after exposure to several varieties of paraffin oil emulsion particles, even if the challenged cells had been pretreated with colchicine of cytochalasin B. (5) Since suramin, which inhibited Mg2+-ATPase, had no effect upon other granulocyte functions such as chemotaxis, superoxide anion generation, or phagocytosis, it is unlikely that the enzyme plays a major role in these functions.     

Interaction of Cd2+ with the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of human erythrocyte ghosts

Treatment of erythrocyte ghosts with micromolar concentrations of Cd2+ results in a noncompetitive inhibition of the calmodulin-dependent (Ca2+ + Mg2+)-ATPase activity. Higher concentrations of Cd2+ are required for inhibition of the (Ca2+ + Mg2+)-ATPase activity of calmodulin-depleted ghosts. The interaction of Cd2+ is time-dependent with an apparent rate constant around 0.12/min. The inhibition is relieved by addition of EGTA with a rate constant around 0.15/min. If Cd2+ is allowed to interact with calmodulin prior to the association of the protein with the ghosts, the inhibition is mainly competitive. The results suggest that the inhibitory effect caused by Cd2+ is due to an interaction with calmodulin. The slow interaction of Cd2+ suggests that calmodulin bound to the (Ca2+ + Mg2+)-ATPase is inaccessible to Cd2+.     

Ionic regulation of adenylate cyclase from the cilia of Paramecium tetraurelia.

The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.     

Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.     

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.     

Vanadate inhibition of the Ca2+-ATPase from human red cell membranes

(1) VO3(-) combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state of the Ca2+-dependent phosphoenzyme. (2) VO3(-) blocks hydrolysis of ATP at the catalytic site. The sites for VO3(-) also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase. (3) The sites for VO39-) show positive interaactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3(-). Although, with less effectiveness, Na2+ and K+ substitutes for K+ whereas Li+ does not. The apparent affinites for Mg24 and K+ for inhibiton by VO3(-) seem to be less than those for activation of the Ca2+-ATPase. (4) Inhibition by VO3(-) is independent of Ca2+ at concentrations up to 50 microM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitiory effect of VO3(-).     

Exaprolol as a modulator of heart sarcolemmal (Na+ + K+)-ATPase. Evidence for interaction with an essential sulfhydryl group in the catalytic centre of the enzyme

Beta-adrenoceptor blocking agents may have, in addition to their primary action, also ancillary effects on the cell membrane. In the present paper the non-specific interaction of exaprolol with the ATPase systems in isolated rat heart sarcolemmal membranes was investigated. When preincubated with sarcolemmal membranes in vitro, exaprolol in concentrations below 10(-4) mol.l-1 had no significant effect on sarcolemmal Mg2+-, Ca2+- and (Na+ + K+)-ATPase activities. At exaprolol concentration of 10(-4) mol.l-1 the Mg2+- and Ca2+-ATPase activities became inhibited whereas the (Na+ + K+)-ATPase activity was markedly stimulated. A kinetic analysis of these interactions revealed a non-competitive inhibition of Mg2+- and Ca2+-ATPase. In the case of (Na+ + K+)-ATPase a synergistic type of stimulation characterized by an exaprolol-induced conversion of an essential sulfhydryl group in the active site of the enzyme to the more reactive [S-] form has been observed thus increasing the affinity of the enzyme to ATP. Exaprolol concentrations exceeding 5 X 10(-4) mol.l-1 induced an overall depression of the investigated enzyme activities.     

Relationship of Plasmalemma Redox Activity to K+ Uptake by Dunaliella salina Cells

The plasmalemma-bond redox system localized within the plasmalemma of unicellular green alga Dunaliella salina was studied. This system oxidized exogenous NADH, increased O2 consumption to 165 % and increased the pH of the external medium, while K+ influx was inhibited. With no NADH added, ferricyanide stimulated K+ uptake about 3 folds. In the presence of exogenous NADH, ferricyanide was rapidly reduced and the external medium was acidified, generating a greater electrochemical proton gradient across the plasmalemma, thus resulting an 6-fold increase of K+ influx. Typical inhibitors of plasmalemma H+-ATPase and redox system inhibited K+ uptake to different extent. That the inhibition of K+ uptake by vanadate could be resumed partly by addition of NADH and ferricyanide indicated that plasmalemma redox system operated in association with the H+-ATPase to exert an influence on K+ transportation. A model was presented in which the implication of two possible redox chains and H+-ATPase in generating an electrochemical potential gradient for protons (△uH+) was discussed.     

Localization of (Ca2+ + Mg2+)-ATPase, Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1.10(-4) M. The sarcolemmal markers, ouabain-sensitive (Na+ +K+)-ATPase and Na+/Ca2+-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na+ pump (K+-p-nitrophosphatase) were not affected. Almost 20% of the (Ca2+ +Mg2+)-ATPase and Ca2+ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27-39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca2+ +Mg2+-ATPase and Ca2+ pump compared to control hearts. (Ca2+ +Mg2+)-ATPase and Ca2+ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class (K1/2 for inhibition approx. 1.5 microM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg2+-ATPase and Ca2+-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

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.