Wu-Hsiung Huang

Ca2+-dependent activities of (Na+ + K+)-ATPase

Abstract Experiments on the effects of varying concentrations of Ca2+ on the Mg2+ + Na+-dependent ATPase activity of a highly purified preparation of dog kidney (Na+ + K+)-ATPase showed that Ca2+ was a partial inhibitor of this activity. When Ca2+ was added to the reaction mixture instead of Mg2+, there was a ouabain-sensitive Ca2+ + Na+-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg2+ + Na+-dependent activity. The apparent affinities of the enzyme for Ca2+ and CaATP seemed to be higher than those for Mg2+ and MgATP. Addition of K+, along with Ca2+ and Na+, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca2+ + Na+ + K+-dependent ATPase was about two orders of magnitude smaller than that of Mg2+ + Na+ + K+-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca2+, Na+, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K+. The enzyme also exhibited a Ca2+ + Na+-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca2+ on Mg2+-dependent activities, nor the Ca2+-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg2+ in all reaction mixtures, it could not be determined whether the apparent Ca2+-dependent activities involved enzyme-substrate complexes containing Ca2+ as the divalent cation or both Ca2+ and Mg2+.

Transport ATPase—the different modes of inhibition of the enzyme by various mercury compounds

Abstract The effects of a number of mercurials on the partial reactions of Na+, K+-ATPase were studied. Results of these studies allow a classification of the mercurials into two groups: selective modifiers of the enzyme and unspecific inhibitors. Thimerosal, ethylmercury and methylmercury were shown to selectively inhibit Na+, K+-ATPase while not affecting K+-NPPase, Na+-ATPase, ADP-ATP exchange reaction and phosphoenzyme formation. In contrast, p-chloromercuribenzoic acid, p-chloromercuriphenylsulfonic acid, mersalyl and mercuric chloride inhibited Na+, K+-ATPase as well as several partial reactions of the enzyme in a parallel manner. The selective modification of Na+, K+-ATPase by compounds such as ethylmercury and methylmercury makes these agents useful probes of the reaction mechanism of the enzyme. Whether this mode of inhibition of the enzyme is related to the mechanism of toxicity of short-chain alkylmercury compounds remains to be determined.

Origins of the different sensitivities of (Na+ + K+)-dependent adenosinetriphosphatase preparations to ouabain

Properties of the Na+, K+-ATPase preparations from rat and dog kidney medullae were compared. The two enzymes, with a 1000-fold difference in ouabain sensitivities, had similar subunit compositions and similar K0.5 values and Hill coefficients for substrates and activators of several catalytic activities; suggesting that the structural differences of the two are limited to the ouabain binding domains. Experiments on the interactions of ouabain, Pi, and Mg2+ with the enzymes showed that the two enzymes differed (a) in their inherent affinities for ouabain; and (b) in that Mg2+ binding increased affinity for ouabain to a greater extent in the dog enzyme than in the rat enzyme.

(Na+ + K+)-ATPase Phosphorylation-dependent cross-linking of the α-subunits in the presence of Cu2+ and o-phenanthroline

In previous studies we had demonstrated that in the presence of 0.25 mM Cu2+ and 1.25 mM o-phenanthroline, cross-linking of the alpha-subunits of Na+ + K+)-dependent adenosine triphosphatase was induced by the addition of Na+ + ATP, and that the formation of the alpha,alpha-dimer was preceded by that of phosphoenzyme. The purpose of the present studies was the further evaluation of the role of phosphoenzyme in the process of cross-linking. Na+ + UTP did not induce cross-linking unless Mg2+ was also added. In contrast, Na+ + ATP-induced cross-linking did not require the addition of Mg2+. The different effects of ATP and UTP in the absence of added Mg2+ could be accounted for by the presence in the enzyme preparation of bound Mg2+ which supported enzyme phosphorylation by ATP but not by UTP. When the enzyme was phosphorylated by Pi, in the presence of Mg2 and ouabain, and the exposed to Cu2+ and o-phenanthroline, the alpha,alpha-dimer was obtained. Under these conditions, Na+ blocked both phosphorylation and cross-linking. These results indicate that it is the formation of phosphoenzyme per se that leads to conformational transitions favorable to cross-linking. They also suggest that Cu2+ and o-phenanthroline participate in the cross-linking reaction, but not in the phosphorylation reactions. In the digitonin-treated enzyme, Na+ and ATP induced the formation of phosphoenzyme, but not that of alpha,alpha-dimer. These findings indicate that in addition to phosphorylation, a proper orientation o alpha-subunits in an oligomer is also necessary for cross-linking.

Transport ATPase of erythrocyte membrane: Sensitivities of Na+,K+-ATPase and K+-phosphatase activities to ouabain

Abstract Cardiac glycosides are inhibitors of Na+,K+-ATPase, and K+-phosphatase activities of the transport enzyme. Previous studies have shown that when the sensitivities of these two activities to ouabain are compared by the addition of varying concentrations of the drug to the assay media, the K+-phosphatase is significantly less sensitive than Na+,K+-ATPase. This work was done to seek an explanation for this phenomenon. 3-O-Methyl-fluorescein phosphate was used as substrate for the continuous fluorimetric assay of K+-phosphatase obtained from human red cells. When ouabain was added to the assay medium, a time-dependent inhibition of K+-phosphatase was observed. The rate of inhibition was also influenced by the order of additions of K+ and ouabain. In view of these results, several enzyme samples exposed to ouabain for varying lengths of time were prepared, and their Na+,K+-ATPase and K+-phosphatase activities were then determined. A good correlation between the extent of inhibition of the two activities was obtained. These results prove that the previously observed discrepancies between the sensitivities of Na+,K+-ATPase and K+-phosphatase to ouabain are due to the different kinetics of drug interaction with the enzyme under the different conditions of the two assays and that once a certain level of ouabain binding to the enzyme is achieved, both activities are equally inhibited.

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca2+ inhibited the Mg2+-dependent and K+-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na+ + K+)-ATPase. In the absence of K+, however, a Mg2+-dependent and Ca2+-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K+-stimulated phosphatase. The Ca2+-stimulated phosphatase, like the K+-stimulated activity, was inhibited by either ouabain or Na+ or ATP. Ouabain sensitivity was decreased with increase in Ca2+, but the K0.5 values of the inhibitory effects of Na+ and ATP were independent of Ca2+ concentration. Optimal pH was 7.0 for Ca2+-stimulated activity, and 7.8-8.2 for the K+-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca2+-stimulated phosphatase is catalyzed by (Na+ + K+)-ATPase; (b) there is a site of Ca2+ action different from the site at which Ca2+ inhibits in competition with Mg2+; and (c) Ca2+ stimulation can not be explained easily by the action of Ca2+ at either the Na+ site or the K+ site.

(Na+ + K+)-ATPase: effects of detergents on the cross-linking of subunits in the presence of Cu2+ and o-phenanthroline.

When (Na+ + K+)-ATPase is reacted with Cu2+ or Cu2+-phenanthroline, cross-linking of the two subunits (alpha and beta) occurs. The major products are alpha,beta- and alpha,alpha-dimers. The alpha,beta-dimer is unstable in the presence of EDTA, but becomes stable when it is first exposed to digitonin or Triton X-100. Conversion of alpha-CU2+-beta to alpha-S-S-beta is suggested. If the enzyme that is pretreated with these detergents is used, only the stable alpha,beta-dimer is obtained, and the formation of alpha,alpha-dimer is inhibited. The data are consistent with alpha 2 beta 2 quaternary structure of the enzyme.

Autoregulation of the phosphointermediate of Na+/K+-ATPase by the amino-terminal domain of the α-subunit

Chymotryptic cleavage of the alpha-subunit of the canine kidney Na+/K(+)-ATPase in the presence of Na+ abolishes ATPase activity and yields an 83 kDa peptide from Ala 267 to the COOH-terminus. To test the proposal that E1 to E2 conformational transition is blocked in this modified enzyme, we have made a detailed comparison of its phosphorylation with that of the native enzyme by ATP. While phosphorylation of alpha is dependent on Na+ and prevented by K+, that of the 83 kDa peptide is modestly stimulated by Na+; and only this stimulation, but not the Na(+)-independent phosphorylation is inhibited by K+. Ouabain, which inhibits alpha-phosphorylation by ATP, activates Na(+)-independent phosphorylation of the 83 kDa peptide by ATP, and inhibits the Na(+)-stimulation of this process. While there is a ouabain-stimulated phosphorylation of alpha by Pi, the 83 kDa peptide is not phosphorylated by Pi with or without ouabain. In its sensitivity to ADP, and insensitivity to K+, the phosphopeptide is similar to the E1P of the native enzyme; however, the spontaneous decomposition rate of the phosphopeptide is orders of magnitude lower than that of the native EP. Na+ has no effect on the spontaneous decomposition of the phosphopeptide; but at high Na+ concentrations (K0.5 = 350 mM) the ADP sensitivity of the phosphopeptide is reduced. The phosphopeptide, like the native EP, is acid-stable, alkaline-labile, and sensitive to hydroxylamine and molybdate. The chymotrypsin-treated enzyme catalyzes an ADP-ATP exchange activity that is stimulated by Na+. The Na(+)-independent part of this exchange, unlike that of the native enzyme, is activated by ouabain. Our findings establish that (a) the phosphorylation process and its control by Na+, K+ and ouabain are autoregulated by the NH2-terminal domain of the alpha-subunit; and (b) the often repeated assumption that the primary role of this domain is in the regulation of E1-E2 transitions is not valid.

Na+, K+-ATPase: (Ca2+ + ouabain)-dependent phosphorylation by Pi.

The active transports of Na’ and K’ are done by Na’,K’-ATPase (EC 3.6.1.3) of the plasma membrane [l]. The enzyme, which requires Mg2+ and is inhibited by ouabain and related cardiac glycosides, is also considered to be the receptor for some, if not all, of the cardiac effects of these drugs [2]. Studies on the interaction of Ca*+ with this enzyme are of special interest because of the long-established interrelation between the effects of Ca*+ and cardiac glycosides on the heart [3]. Inhibition of enzyme activity by Ca*+, and the effects of Ca*’ on enzyme phosphorylation by ATP have been studied [2,4,5]. Na’,K’-ATPase is also phosphorylated by Pi in the presence of either Mg*+ or Mg*+ and ouabain [6-91. Here we report that the enzyme may also be phosphorylated by Pi in the presence of Ca*+, but that this process has an obligatory requirement for ouabain. The maximal level of phosphoenzyme formed with Pi + Ca** + ouabain is half of that obtained with Pi + Mg*+ + ouabain. The maximal level of bound ouabain is the same under both conditions. The findings provide support for Ca*+ + ouabaininduced half-of-the-sites reactivity in the oligomeric enzyme.

Na+,K+,-ATPase: ligand-induced alterations in subunit crosslinking

(Na’ + K’)-dependent adenosinetriphosphatase (Na’,K’-ATPase EC 3.6.1.3) is the enzyme that performs the energy-dependent transports of Na’ and K’ across the plasma membrane [ 11. The purified enzyme contains two polypeptides in association with the lipids of the membrane. The a-subunit with mol. wt -100 000 is the catalytic unit of the enzyme, and the P-subunit with mol. wt -40 000 is a glycoprotein of uncertain role. To explore the quaternary structure of the enzyme, several studies on the chemical crosslinking of the enzyme subunits have been attempted, and the results have been interpreted in support of an CQ& structure of the enzyme [2-61. Here we report that the formation of a crosslinked cy,cy-dimer in the presence of Cu*‘-phenanthroline is influenced by certain physiological ligands of the enzyme. The results provide little new information concerning the quaternary structure of the enzyme. They do clearly show, however, the usefulness of crosslinking reagents as novel conformational probes of Na’,K’-ATPase.