Studies on the relationship between glycolysis and (Na++K+)-ATPase in cultured cells

In several tissues a coupling between glycolysis and (Na⁺+K⁺)-ATPase has been observed. We report here studies on the coupling of glycolysis and (Na⁺+K⁺)-ATPase in Rous-transformed hamster cells and Ehrlich ascites tumor cells. The rate of (Na⁺+K⁺)-ATPase was estimated by the initial rate of ouabain-sensitive K⁺ influx after K⁺ reintroduction to K⁺-depleted cells. Experiments were performed with cells producing ATP via oxidative phosphorylation alone (i.e., lactate sole substrate), glycolysis alone (i.e., glucose as substrate in the absence of oxygen or with antimycin A), or glycolysis and oxidative phosphorylation (i.e., glucose as substrate in the presence of oxygen). The cells produced ATP at approximately the same rate under all of these conditions, but the initial rate of K⁺-influx was approx. 2-fold higher when AtP was produced from glycolysis. Changes in cell Na⁺ due to other transport processes related to glycolysis, such as Na⁺-H⁺ exchange, Na⁺-glucose cotransport, and K⁺-H⁺ exchange were ruled out as mediators of this effect on (Na⁺+K⁺)-ATPase. These data suggest that glycolysis is more effective than oxidative phosphorylation in providing ATP to (Na⁺+K⁺)-ATPase to these cultured 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).     

The sodium cycle. II. Na+-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na+ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na+-motive respiratory chain of V. alginolyticus. Na+ loaded cells incubated in a K+ or Li+ medium fail to synthesize ATP in response to lactate addition. The addition of Na+ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na+ level. Neither lactate oxidation nor delta psi generation coupled with this oxidation is increased by external Na+ in the Na+-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse delta pNa, i.e., [Na+]in greater than [Na+]out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K+-loaded cells incubated in a high Na+ medium, i.e., when delta pNa of the proper direction [( Na+]in less than [Na+]out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K+ medium (delta pNa is low) is decreased by CCCP even without monensin. Artificial formation of delta pNa by adding 0.25 M NaCl to the K+-loaded cells (Na+ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na+ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na+ instead of H+ as the coupling ion.     

Regulation of rat brain (Na+ +K+)-ATPase activity by cyclic AMP

The interaction between the (Na+ +K+)-ATPase and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5'-AMP, cyclic GMP or 5'-GMP, could inhibit the (Na+ +K+)-ATPase enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of (Na+ +K+)-ATPase enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854-3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in (Na+ +K+)-ATPase activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in (Na+ +K+)-ATPase enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of (Na+ +K+)-ATPase, resulted in a decrease in overall (Na+ +K+)-ATPase activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the (Na+ +K+)-ATPase has no effect on (Na+ +K+)-ATPase activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the (Na+ +K+)-ATPase enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the (Na+ +K+)-ATPase was unaffected.     

ATP-producing and consuming processes of Ehrlich mouse ascites tumor cells in proliferating and resting phases.

The extents of ATP-yielding and consuming processes in Ehrlich mouse ascites tumor cells during the proliferating and resting growth phase were compared. In the resting phase the total ATP production was decreased by one-third. The ATP supply by oxidative phosphorylation was drastically reduced, whereas the rate of glycolysis stayed nearly constant. All ATP-consuming processes investigated, i.e., protein turnover, Na+/K(+)-ATPase, Ca2(+)-ATPase, and RNA synthesis, were decreased proportionally with the total ATP consumption.     

Na+-Na+ exchange mediated by (Na+ + K+)-ATPase reconstituted into liposomes. Evaluation of pump stoichiometry and response to ATP and ADP

(Na+ + K+)-ATPase from shark rectal glands reconstituted into lipid vesicles and oriented inside out catalyses an ouabain-sensitive Na+-Na+ exchange in the absence of intravesicular K+ when ATP is added extravesicularly. Intravesicular ouabain inhibited the exchange completely. This was also the case with digitoxigenin added to the vesicles. Intravesicular oligomycin inhibited the Na+-Na+ exchange partly in a fashion which was ATP dependent. The exchange is accompanied by a net hydrolysis of ATP with an apparent Km of 2.5 microM. ADP was found to give no stimulation of the Na+-Na+ exchange, contrarily, ADP inhibited the ATP-dependent exchange of Na+ both at optimal and supraoptimal ATP concentrations. When initial influx and efflux of 22Na was measured and the hydrolysis of ATP concomitantly determined a coupling ratio of 2.8:1.3:1 was found, i.e. 2.8 moles of Na+ were taken up (cellular efflux) and 1.3 moles of Na+ extruded (cellular influx) for each mole of ATP hydrolyzed. The electrogenic Na+-Na+ exchange generated a transmembrane potential which was measured with the fluorescent probe ANS (8-anilino-1-naphthalenesulfonic acid) to be 60 mV positive inside the liposomes (extracellular).     

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.     

Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity

N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+)-ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some total and partial reactions of Na+/K+-ATPase using ATP and acetyl phosphate as a substrate

Acetyl phosphate, as a substrate of (Na+ + K+)-ATPase, was further characterized by comparing its effects with those of ATP on some total and partial reactions carried out by the enzyme. In the absence of Mg2+ acetyl phosphate could not induce disocclusion (release) of Rb+ from E2(Rb); nor did it affect the acceleration of Rb+ release by non-limiting concentrations of ADP. In K+-free solutions and at pH 7.4 sodium ions were essential for ATP hydrolysis by (Na+ + K+)-ATPase; when acetyl phosphate was the substrate a hydrolysis (inhibited by ouabain) was observed in the presence and absence of Na+. In liposomes with (Na+ + K+)-ATPase incorporated and exposed to extravesicular (intracellular) Na+, acetyl phosphate could sustain a ouabain-sensitive Rb+ efflux; the levels of that flux were similar to those obtained with micromolar concentrations of ATP. When the liposomes were incubated in the absence of extravesicular Na+ a ouabain-sensitive Rb+ efflux could not be detected with either substrate. Native (Na+ + K+)-ATPase was phosphorylated at 0 degrees C in the presence of NaCl (50 mM for ATP and 10 mM for acetyl phosphate); after phosphorylation had been stopped by simultaneous addition of excess trans-1,2-diaminocyclohexane-N,N,N',N' tetraacetic acid and 1 M NaCl net synthesis of ATP by addition of ADP was obtained with both phosphoenzymes. The present results show that acetyl phosphate can fuel the overall cycle of cation translocation by (Na+ + K+)-ATPase acting only at the catalytic substrate site; this takes place via the formation of phosphorylated intermediates which can lead to ATP synthesis in a way which is indistinguishable from that obtained with ATP.     

Distinction between the intermediates in Na+-ATPase and Na+,K+-ATPase reactions. II. Exchange and hydrolysis kinetics at micromolar nucleotide concentrations

The ATP hydrolysis rate and the ADP-ATP exchange rate of (Na+ + K+)-ATPase from ox brain were measured at 10 microM Mg2+free and at micromolar concentrations of free ATP and ADP. (1) In the absence of K+, substrate inhibition of the hydrolysis rate was observed. It disappeared at low Na+ and diminished at increasing concentrations of ADP. This was interpreted in terms of free ATP binding to E1P. In support of this interpretation, free ATP was found to competitively inhibit ADP-ATP exchange. (2) In the presence of K+, substrate activation of the hydrolysis rate was observed. Increasing (microM) concentrations of ADP did not give rise to competitive inhibition in contrast to the situation in the absence of K+ (cf. 1, above). This was interpreted to show that at micromolar substrate, some low-affinity, high-turnover Na+ + K+ activity is possible, provided the Mg2+ concentration is low. (3) While small concentrations of K+ increased the hydrolysis rate (cf. 2) they decreased the rate of ADP-ATP exchange. To elucidate this phenomenon, parallel measurements of exchange and hydrolysis rates were performed over a wide range of ATP and ADP concentrations, with and without K+. If, in the presence and absence of K+, ADP (and ATP competing) are binding to the same phosphorylated intermediate for the backward reaction, it places quantitative restrictions on the ratio of rate constants with and without K+. The results did not conform to these restrictions, and the discrepancy is taken as evidence for the necessity for a bicyclic scheme for the action of the (Na+ + K+)-ATPase. (4) An earlier statement concerning the nature of the phosphoenzyme obtained in the presence of Na+ and K+ is amended.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain (Na+ + K+)-ATPase with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1) Na+ + Mg2+ + ATP, (2) Mg2+, Mg2+ + K+, K+ and none, (3) Na+ + Mg2+, Na+, K+ + Na+ and Na+ + K+ + Mg2+, (4) Mg2+ + K+ + ATP, K+ + ATP and Mg2+ + ATP, (5) K+ + Na + + ATP, Na+ + ATP, Na+ + K+ + Mg2+ + ATP and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition (KS0.5) were 3 mM, 0.13 mM and 4 MicroM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i. e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of (Na+ + K+)- ATPase proceeds via at least four kinds of enzyme form (E1, E2, E1 . nucleotide and EP), which all have different conformations.     

Kinetic studies on the (Na+ + K+)-dependent ATPase evidence for coexisting sites for Na+, K+ and Mg2+

Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+ -ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.     

Effect of external ATP on the plasma membrane permeability and (Na+ +K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na+ +K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na+ +K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.     

Kinetics of (Na+ + K+)-ATPase: analysis of the influence of Na+ and K+ by steady-state kinetics

The influence of Na+ and K+ on the steady-state kinetics at 37 degrees C of (Na+ + K+)-ATPase was investigated. From an analysis of the dependence of slopes and intercepts (from double-reciprocal plots or from Hanes plots) of the primary data on Na+ and K+ concentrations a detailed model for the interaction of the cations with the individual steps in the mechanism may be inferred and a set of intrinsic (i.e. cation independent) rate constants and cation dissociation constants are obtained. A comparison of the rate constants with those obtained from an analogous analysis of Na+-ATPase kinetics (preceding paper) provides evidence that the ATP hydrolysis proceeds through a series of intermediates, all of which are kinetically different from those responsible for the Na+-ATPase activity. The complete model for the enzyme thus involves two distinct, but doubly connected, hydrolysis cycles. The model derived for (Na+ + K+)-ATPase has the following properties: The empty, substrate free, enzyme form is the K+-bound form E2K. Na+ (Kd = 9 mM) and MgATP (Kd = 0.48 mM), in that order, must be bound to it in order to effect K+ release. Thus Na+ and K+ are simultaneously present on the enzyme in part of the reaction cycle. Each enzyme unit has three equivalent and independent Na+ sites. K+ binding to high-affinity sites (Kd = 1.4 mM) on the presumed phosphorylated intermediate is preceded by release of Na+ from low-affinity sites (Kd = 430 mM). The stoichiometry is variable, and may be Na:K:ATP = 3:2:1. To the extent that the transport properties of the enzyme are reflected in the kinetic ATPase model, these properties are in accord with one of the models shown by Sachs ((1980) J. Physiol. 302, 219-240) to give a quantitative fit of transport data for red blood cells.     

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.     

Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphate and the effects of enzyme inhibitors.

The involvement of membrane (Na+ + K+)-ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na+ + K+)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K+ required the simultaneous presence of Na+. Ouabain, a specific inhibitor of (Na+ + K+)-ATPase, significantly inhibited the (Na+ + K+)-stimulation of respiration. These observations suggest that the (Na+ + K+)-stimulation of brain slice respiration is related to ADP production as a result of (Na+ + K+)-ATPase activity. However, ouabain also inhibited non-K+ -stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na+ + K+)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na+ + K+)-ATPase inhibition and acts additionally by increasing the intracellular Na+ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na+ + K+)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na+ and K+ media and that in choline, K+ media. Studies were performed with two (Na+ + K+)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na+ + K+)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na+ + K+)-ATPase and related respiration, but chlorpromazine failed to alter either (Na+ + K+)-ATPase activity or related respiration.     

The sided action of Na+ and of K+ on reconstituted shark (Na+ + K+)-ATPase engaged in Na+-Na+ exchange accompanied by ATP hydrolysis. I. The ATP activation curve

The ATP hydrolysis dependent Na+-Na+ exchange of reconstituted shark (Na+ + K+)-ATPase is electrogenic with a transport stoichiometry as for the Na+-K+ exchange, suggesting that translocation of extracellular Na+ is taking place via the same route as extracellular K+. The preparation thus offers an opportunity to compare the sided action of Na+ and K+ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K+. With Na+ but no K+ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K+ in concentrations of 50 microM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K+ also gives biphasic ATP-activation curves, however, only when the K+ concentration is 50 mM or higher (Na+ + K+ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E2(Na2+) and E2(K2+), respectively, and in which the dephosphorylation of E2-P is rate limiting in the presence of Na+ (but no K+) extracellular, whereas in the presence of extracellular K+ it is the deocclusion of E2(K2+) which is rate limiting.     

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding.

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Pb2+ and imidazole-activated phosphorylation by ATP of (Na+ + K+)-ATPase

In order to study whether Pb2+ and imidazole increase the ATP phosphorylation level of (Na+ + K+)-ATPase by the same mechanism, the effects of both compounds on phosphorylation and dephosphorylation reactions of the enzyme have been studied. Imidazole in the presence of Mg2+ increases steady-state phosphorylation of (Na+ + K+)-ATPase by decreasing, in a competitive way, the K+-sensitivity of the formed phospho-enzyme (E-P . Mg). If Pb2+ is present during phosphorylation, the rate of phosphorylation increases and a K+- and ADP-insensitive phosphointermediate (E-P . Pb) is formed. Pb2+ has no effect on the K+-sensitivity of E-P . Mg and EDTA is unable to affect the K+-insensitivity of E-P . Pb. These effects indicate that Pb2+ acts before or during phosphorylation with the enzyme. Binding of Na+ to E-P . Pb does not restore K+-sensitivity either. However, increasing Na+ during phosphorylation in the presence of Pb2+ leads to formation of the K+-sensitive intermediate (E-P . Mg), indicating that E-P . Pb is formed via a side path of the Albers-Post scheme. ATP and ADP decrease the dephosphorylation rate of both E-P . Mg and E-P . Pb. Above optimal concentration, Pb2+ also decreases the steady-state phosphorylation level both in the absence and in the presence of Na+. This inhibitory effect of Pb2+ is antagonized by Mg2+.