Phosphorylated intermediates of Na,K-ATPase proteoliposomes controlled by bilayer cholesterol. Interaction with cardiac steroid

Fragmental Na,K-ATPase from the electric eel forms three phosphorylated intermediates (EP) with MgATP and Na+: ADP-sensitive K+-insensitive EP (E1P), ADP- and K+-sensitive EP (E*P), and K+-sensitive ADP-insensitive EP (E2P). The EP composition varied with the Na+ concentration. In the reconstituted Na,K-ATPase proteoliposomes (PL), the EP composition of the inside-out form was controlled not only by the intravesicular (extracellular) Na+ concentration, but also by the temperature and the cholesterol content of the lipid bilayer. When the lipid bilayer of PL contained less than 30 mol % cholesterol, the E*P content did not change significantly while the E2P content increased with an elevation in temperature (3-20 degrees C). In contrast, when the lipid bilayer contains more than 35 mol % cholesterol, the E*P content increased while the E2P content stayed less than 10% under the same temperature change. These observations suggest that a high cholesterol content in the lipid bilayer interferes with the E*P to E2P conversion. This cholesterol effect was reversed by ionophores (monensin, nigericin, and A23187). Therefore, E1P-rich EP, E*P-rich EP, or E2P-rich EP could be obtained in the PL under a constant Na+ concentration by using various concentrations of cholesterol and ionophores. The reaction between the proteoliposomal EPs and digitoxigenin (lipid-soluble cardiac steroid) occurred in a single turnover, thereby avoiding unphysiologically high Na+ concentrations. The increase in the ADP- and K+-insensitive EP, which indicated formation of the digitoxigenin-Na,K-ATPase complex, was equivalent to the decrease in the E*P under six different sets of conditions, without any significant change in the E1P and E2P contents. This result indicated that E*P is the active intermediate of the Na,K-ATPase for cardiac steroid binding. Although the E2P has been thought to be the active form for binding, it cannot bind with the cardiac steroid in the presence of Na+ and in the absence of free Mg2+.     

Cytoplasmic K+ effects on phosphoenzyme of Na,K-ATPase proteoliposomes and on the Na+-pump activity

Three phosphorylated reaction intermediates (EP) of Na,K-ATPase, and ADP-sensitive K+-insensitive EP (E1P), an ADP- and K+-sensitive EP (E*P), and a K+-sensitive ADP-insensitive EP (E2P), have been discovered at present. By using Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme, we found in this study that E*P existed even in the presence of K+ on both sides of the PL and that there was a sidedness difference in K+ sites between E*P and E2P. Cytoplasmic K+ (K+cyt) accelerated the conversion of E*P to E2P but did not dephosphorylate the E2P. Although the extracellular K+ accelerated the dephosphorylation of E2P, it did not interact with E*P directly. This K+cyt effect was also verified by the activation of Na+-pump in the Na+-K+ exchange mode. In the presence of K+cyt, both the ATP hydrolysis and Na+ uptake rates of the PL containing K+ inside vesicles increased sigmoidally with the concentrations of ATP and cytoplasmic Na+ (Na+cyt). However, in the absence of K+cyt, these Na+-pump reactions in PL containing K+ inside vesicles had only a hyperbolic curve. These results imply that the E*P to E2P conversion is one of the rate-limiting steps of the Na+-pump in the presence of a high concentration of ATP and that K+cyt may control this reaction step by enhancing the conversion rate of E*P to E2P.     

Acetyl phosphate can act as a substrate for Na+ transport by (Na+ + K+)-ATPase

Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate.     

ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase

In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP-sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This unusual phenomenon can be explained by the formation of three phosphoenzymes: ADP-sensitive K+-insensitive (E1P), K+-sensitive ADP-insensitive (E2P), and ADP- and K+-sensitive (E*P) phosphoenzymes, as proposed by N?rby et al. (N?rby, J. G., Klodos, I., and Christiansen, N. O. (1983) J. Gen. Physiol. 82, 725-757). By applying a simple approximation method for the assay of E1P, E*P, and E2P, it was found that the phosphorylation of the enzyme was much faster than the conversion among each EP and the phosphoenzyme changed as E1NaATP----E1P----E*P----E2P. In the fragmental eel enzyme, the step of E*P to E2P was much slower than the step of E1P to E*P. In the steady state, the E1P was predominant above 400 mM Na+, whereas E*P and E2P were predominant between 60 and 300 mM Na+ and below 60 mM Na+, respectively. The characteristic difference of the eel enzyme from the beef brain enzyme and probably from the kidney enzyme seems to be that the dissociation constant of Na+ on the E1P-E*P equilibrium is higher than that on the E*P-E2P. The E*P and E1P both interacted with ADP to form ATP without formation of inorganic phosphate in the absence of free Mg2+. In the Na,K-ATPase proteoliposomes, the vesicle membrane interfered with the conversion of E1P to E2P, especially the change of E1P to E*P, and furthermore, the E1P content increased. This barrier effect was partially counteracted by monensin or carbonyl cyanide m-chlorophenylhydrazone. Oligomycin reacted with E1P and probably with E*P, therefore inhibiting their conversion to E2P and interaction with K+.     

Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+

To determine the biochemical events of Na+ transport, we studied the interactions of Na+, Tris+, and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain. The enzyme was phosphorylated by incubation at 0 degrees C with 1 mM Mg2+, 25 microM [32P]ATP, and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+, and (4) 2.5 mM ADP plus Na+ up to 600 mM. In dephosphorylation types 2-4, the curves were bi- or multiphasic. "ADP-sensitive EP" and "K+-sensitive EP" were determined by extrapolation of the slow phase of the curves to the ordinate and their sum was always larger than Etotal. These results required a minimal model consisting of three consecutive EP pools, A, B, and C, where A was ADP sensitive and both B and C were K+ sensitive. At high [Na+], B was converted rapidly to A (type 4 experiment). The seven rate coefficients were dependent on [Na+], [Tris+], and [K+], and to explain this we developed a comprehensive model for cation interaction with EP. The model has the following features: A, B, and C are equilibrium mixtures of EP forms; EP in A has two to three Na ions bound at high-affinity (internal) sites, pool B has three, and pool C has two to three low-affinity (external) sites. The putative high-affinity outside Na+ site may be on E2P in pool C. The A leads to B conversion is blocked by K+ (and Tris+). We conclude that pool A can be an intermediate only in the Na-ATPase reaction and not in the normal operation of the Na,K pump.     

Selective inhibition by ionophore A23187 of the enzyme isomerization in the catalytic cycle of Na+, K+ -ATPase

The effect of an ionophore A23187 on the purified Na+,K+-ATPase from the outer medulla of pig kidney was investigated. When the enzyme was pretreated with A23187 in the presence of Na+ and K+, the ATPase activity was inhibited almost completely. When the pretreatment was performed in the presence of Na+ and absence of K+, formation of the phosphoenzyme (EP) from ATP was only slightly retarded. The steady state level of EP thus formed was not altered, but EP decomposition was strongly inhibited. Under these conditions, the accumulated EP was sensitive to ADP and insensitive to K+. On the other hand, when the pretreatment was performed in the absence of Na+ and presence of K+, EP formation following simultaneous addition of Na+ and ATP was extremely slow, but the steady state level of EP was not substantially altered. When the pretreatment was performed in the absence of Na+ and presence of K+, EP formation from Pi was unaffected, and the EP formed was in rapid equilibrium with Pi of the medium. These results demonstrate that A23187 selectively inhibits isomerization of the enzyme between the high Na+ and low K+ affinity form and the low Na+ and high K+ affinity form in the catalytic cycle, whether or not the enzyme is phosphorylated. This inhibition is quite similar to the A23187-induced inhibition of the enzyme isomerization in the catalytic cycle of the Ca2+ -ATPase from sarcoplasmic reticulum (Hara, H., and Kanazawa, T. (1986)J. Biol. Chem.261, 16584-16590). These findings suggest that some common mechanism, which is involved in the enzyme isomerization, between these two transport ATPases is strongly disturbed by A23187.     

Effects of sodium and potassium ions on the elementary steps in the reaction of Na+-K+-dependent ATPase1.

The effects of Na+ and K+ ions on the elementary steps in the reaction of Na+-K+-dependent ATPase (EC 3.6.1.3) were investigated in 0.5-600mM NaCL and 0-10mM KCL, at a fixed concentration (1mM) OF MgCL2, AT PH 8.5 and at 15 degrees. The data were analyzed on the basis of the reaction mechanism in which a phosphorylated intermediate, E ADP P (abbreviated as EP), is formed via two kinds of enzyme-substrate comples, E1ATP and E2ATP, and EP is in equilibrium with E2ATP, and is hydrolyzed to produce P1 and ADP. The following results were obtained: 1. The rate od E2ATP-formation, vf, increased with increase in the Na+ concentration, reached a maximum level, and then decreased with further increase in the Na+ concentration at various K+ concentrations. The value of vf was given as (see article). 2. The reciprocal of the equilibrium constants, K2, of the step E1ATPEQUILIBRIUM E ADP P in the presence of low concentrations of Na+ was larger than that in the presence of high concrntrations of Na+, indicating that the equilibrium shifted markedly toward E2ATP at low concentrations of Na+. The relation of K3 with Na concentration was rather complicated on varying the concentration of K+. However, generally speaking, it increased with increase in the K+ concentration. 3. The decomposition of EP was markedly activated by even low concentrations of K+, and inhibited by high concentrations of Na+. The inhibition by Na+ was partially suppressed by K+. The rate constant of EP-decomposition, vo/(EP), was given by (see article) where (vo/(EP) K+EQUALS0 was the value of vo/[EP] in the absence of K+.     

New evidence for ATP binding induced catalytic subunit interactions in pig kidney Na/K-ATPase

Pig kidney Na/K-ATPase preparations showed a positive cooperative effect for pNPP in Na-pNPPase activity. Measurements of the Na-pNPPase activity, Na-ATPase activity and the accumulation of phosphoenzyme (EP) under conditions of pNPP saturation showed several different ATP affinities. The presence of pNPP reduced both the maximum amount of EP and Na-ATPase activity to half showing a value of 4 and a 3,700-fold reduced ATP affinity for EP formation, and a 7 and 1,300-fold reduced affinity for Na-ATPase activity. The presence of low concentrations of ATP in the phosphorylation induced a 2-fold enhancement in Na-pNPPase activity despite a reduction in available pNPP sites. However, higher concentrations of ATP inhibited the Na-pNPPase activity and a much higher concentration of ATP increased both the phosphorylation and Na-ATPase activity to the maximum levels. The maximum Na-pNPPase activity was 1.7 and 3.4-fold higher without and with ATP, respectively, than the maximum Na-ATPase activity. These data and the pNPP dependent reduction in both Na-ATPase activity and the amount of enzyme bound ATP provide new evidence to show that ATP, pNPP and ATP with pNPP, respectively, induce different subunit interactions resulting a difference in the maximum Na(+)-dependent catalytic activity in tetraprotomeric Na/K-ATPase.     

Properties of the conversion of an enzyme-ATP complex to a phosphorylated intermediate in the reaction of Na+-K+-dependent ATPase1.

Previously, we proposed the following reaction machanism for the transport ATPase (EC 3.6.1.3) reaction in the presence of high concentrations of Mg2+ and Na+:(see article). Some kinetic and thermodynamic properties of steps 3 and 4 were investigated, and the following results were obtained. 1. When the reaction was started by adding ATP to the enzyme in the presence of 50 mM Na+ and 0.5 mM K+ or in the presence of 50mM Na+ and 0.5mM Rb+, the amount of E ADP P increased with time and maintained a constant level after reaching a maximum. We could not observe the initial burst of EP formation, which was observed by Post er al. in the presence of 8 mM Na+ and 0.01 mM Rb+. 2. The existence of quasi-equilibrium between E2ATP and E ADP P in the presence of low concentrations of Na+ was suggested by the fact that the values of the reciprocal of the equilibrium constant, K3 of step 3 obtained by the following three methods were almost the same. a) The value of 1+K3 was estimated from the ratio of vo/[EP] to kd, where vo is the rate of ATP hydrolysis in the steady state, [EP] the concentration of EP, and kd the first-order rate constant of EP disappearance after stopping EP formation. b) This value was also calculated from the ratio of the amount of P1 liberated to that of decrease in EP after stopping EP formation. c) The value of K3 was also calculated from the initial rapid decrease in EP on adding K+ and EDTA, assuming that the rapid decrease was due to a shift of the equilibrium toward E2ATP on adding K+. For example, the value of K3 with 10mM NaCL and 0.5mM KCL was 7--11. Although ATP formation due to a shift of the equilibrium toward E2ATP by a K+ jump in the presence of a low concentration of Na+ was observed at 0 degrees, the amount of ATP formed by a K+ jump at 15 degrees was less than the value expected from the shift of the equilibrium. 3. The values of delta H degrees and delta S degrees of step 3 were estimated in the presence of a sufficient amount of Na+ and in the absence of K+. They were +4--+5 kcal mole minus 1 and +15--+16 entropy units mole minus1, respectively. On the basis of kinetic studies of the elementary steps and the overall reaction of Na+-K+-dependent ATPase [EC 3.6.1.3], we (1--4) showed that a phosphorylated intermediate, EP, is formed via two kinds of enzyme-substrate complex, E1ATP and E2ATP, that the EP is in K+-dependent quasi-equilibrium with E2ATP, and that in the presence of high concentration of Mg2+, EP is in a high-energy state and contains bound ADP, E ADP P.(see article).     

Transient-state kinetics of the ADP-insensitive phosphoenzyme in sarcoplasmic reticulum: implications for transient-state calcium translocation

The kinetics of formation of the ADP-sensitive (EP) and ADP-insensitive (E*P) phosphoenzyme intermediates of the CaATPase in sarcoplasmic reticulum (SR) were investigated by means of the quenched-flow technique. At 21 degrees C, addition of saturating ADP to SR vesicles phosphorylated for 116 ms with 10 microM ATP gave a triphasic pattern of dephosphorylation in which EP and E*P accounted for 33% and 60% of the total phosphoenzyme, respectively. Inorganic phosphate (Pi) release was less than stoichiometric with respect to E*P decay and was not increased by preincubation with Ca2+ ionophore. The fraction of E*P present after only 6 ms of phosphoenzyme formation was similar to that at 116 ms, indicating that isomerization of EP to E*P occurs very rapidly. Comparison of the time course of E*P formation with intravesicular Ca2+ accumulation measured by quenching with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid + ADP revealed that Ca2+ release on the inside of the vesicle was delayed with respect to E*P formation. Since Ca2+ should dissociate rapidly dissociation from the low-affinity transport sites, these results suggest that Ca2+ remains "occluded" after phosphoenzyme isomerization and that a subsequent slow transition controls the rate of Ca2+ release at the intravesicular membrane surface. Analysis of the forward and reverse rate constants for the EP to E*P transition gave an expected steady-state distribution of phosphoenzymes strongly favoring the ADP-insensitive form. In contrast, the observed ratio of EP to E*P was about 1:2. To account for this discrepancy, a mechanism is proposed in which stabilization of the ADP-sensitive phosphoenzyme is brought about by a conformational interaction between adjacent subunits in a dimer.     

Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles

Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.     

5-Iodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover

The fluorescence of (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein was studied under turnover conditions. At 4 degrees C the hydrolysis of ATP is slowed sufficiently to permit study of the effects of Na+, K+, and ATP on the steady-state intermediates. With Na+ and Mg2+ (Na-ATPase conditions), addition of ATP produces a 7% drop in signal that reverts back to the initial, high fluorescence after a steady state of several minutes. K-sensitive phosphoenzyme is formed under these conditions, indicating that the fluorescence signal during the steady state is associated with E2P. Under (Na,K)-ATPase conditions (Na+, K+, Mg2+), micromolar ATP produces a steady-state signal that is 25% lower than the initial fluorescence, with no detectable phosphoenzyme formed. This low-fluorescence intermediate, which is also formed by adding K+ to enzyme in the Na-ATPase steady state described above, resembles the state produced by adding K+ directly to enzyme under equilibrium conditions, i.e. E2K. The K0.5(K+) for the fluorescence decrease and for keeping the enzyme dephosphorylated are nearly identical, indicating that the fluorescence change accompanies K+-dependent dephosphorylation. High ATP increases the steady-state fluorescence during the (Na,K)-ATPase reaction; while oligomycin produces still another steady-state fluorescent intermediate. These last two intermediates may be associated with the formation of E2P and E1P, respectively.     

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.     

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.     

Uncoupling the red cell sodium pump by proteolysis

In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis of the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This was evident both in the absence of intravesicular alkali cations when Na-ATPase was compared to ATP-dependent 22Na+ influx, and in the presence of K+ when Na+/K+ exchange was compared to (Na+ + K+)-activated ATPase. This differential loss in pump versus hydrolysis was observed also when the activities of only intact, non-leaky vesicles were compared and therefore reflects intramolecular uncoupling rather than nonspecific leakage. Although oligomycin and thimerosal, like trypsin and chymotrypsin, inhibit the enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) of Na,K-ATPase, at least as it exists in situ, alters the reaction sequence with respect to the normal ordered mechanism. Accordingly, cytoplasmic Na+ and extracellular K+ bind to the enzyme, stimulate phosphorylation (ATP + E1----E1P + ADP) and dephosphorylation (E2P----E2 + Pi), respectively, but each is then released to the same side from which it had bound; presumably release occurs prior to the conformational transitions of E1P to E2P and E2 to E1. This conclusion is supported by experiments showing that, ar micromolar ATP concentration, the hydrolytic activity (Na-ATPase) of the trypsinized but not the unmodified enzyme is stimulated by K+, consistent with earlier experiments (Hegyvary, C., and Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) showing that the K X E2 to K X E1 transition is slower than the E2 to E1 transition.     

Difference between neuronal and nonneuronal (Na+ + K+)-ATPases in their conformational equilibrium

Several experiments were carried out to study the difference between two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase in the conformational equilibrium. Rat brain (Na+ + K+)-ATPase was much more thermolabile than the kidney enzyme. Both enzymes were protected from heat inactivation not only by Na+ and K+, but also by choline in varying degrees, though there was a difference between the two enzymes in the protection by the ligands. The brain enzyme was partially protected from N-ethylmaleimide (NEM) inactivation by both Na+ and K+, but the effects of the ligands on NEM inactivation of the kidney enzyme were more complex. Though ligands differentially affected the thermostability and NEM sensitivity of the two enzymes, the effects were not simply related to the conformational states. The sensitivity of phosphoenzyme (EP) formed in the presence of ATP, Na+, and Mg2+ to ADP or K+ and K+-p-nitrophenyl phosphatase (pNPPase) was then studied as a probe of the differences in the conformational equilibrium between the two isozymes. The EP of the brain enzyme was partially sensitive to ADP, while those of the heart and kidney enzymes were not. At physiological Na+ concentrations the percentages of E1P formed by the brain and kidney enzymes were determined to be about 40-50 and 10-20% of the total EP, respectively. The hydrolytic activity of pNPP in the presence of Li+, a selective activator at catalytic sites of the reaction, was much higher in the kidney enzyme than in the brain enzyme. The inhibition of K+-stimulated pNPPase by ATP and Na+ was greater in the latter enzyme than in the former. These results suggest that neuronal and nonneuronal (Na+ + K+)-ATPases differ in their conformational equilibrium: the E1 or E1P may be more stable in the alpha(+) than in the alpha during the turnover, and conversely the E2 or E2P may be more stable in the latter than in the former.     

Na+ATPase of the mammalian erythrocyte membrane. Reversibility of phosphorylation at 0 degrees.

When human erythrocyte membranes are phosphorylated with a very low concentration of [gamma-32P]ATP (0.02 muM) at 0 degrees, and then EDTA is added, rapid disappearance of the phosphoenzyme intermediate of Na+ATPase is observed. The initial rapid phase of phosphoenzyme disappearance is, for the most part, not associated with P1 release and its rate constant, kD, is severalfold greater than the ratio of Na+ATPase activity to phosphoenzyme intermediate, v:EP, at steady state. It is concluded that this rapid disappearance of phosphoenzyme is due to resynthesis of ATP via reversal of phosphorylation. In contrast, rapid reversal is not observed when excess nonradioactive ATP is added to reduce E32P formation, provided Mg2+ is present; however, K+ added with the ATP stimulates reversal. Rapid reversal following EDTA addition is unlikely also when higher ATP concentrations (greater than or equal to 10(-6) M) are used to phosphorylate the enzyme since, at higher ATP, kD congruent to v:EP. The results are compatible with the concept that the Na+ATPase enzyme is composed of two or more catalytic subunits, in which ATP at one catalytic site can regulate the reactivity at another site.     

Characterization of the ATP-promoted aspect of Na+-Ca2+ exchange present in squid retinal nerve axolemma

Using an in vitro system which consists of an axolemma-rich vesicle fraction prepared from squid retinal nerve fibers, an Na+-Ca2+ exchange process has been characterized and appears identical with that reported in squid giant axon. This exchange is absolutely dependent on the establishment of an Na+ gradient, shows monovalent and divalent cation specificity and is highly sensitive to monensin, A23187 and valinomycin but not to ouabain, digitoxigenin, vanadate, pentylenetetrazole, tetrodotoxin or tetraethylammonium. Furthermore, it was found that the exchange process is enhanced by the addition of ATP. This ATP-promoted aspects of Na+-Ca2+ exchange shares many similar characteristics with Na+-Ca2+ ATP hydrolysis and may indicate a common mechanism for both activities via a protein phosphorylation-dephosphorylation event.     

Effects of divalent cations and pH on amiloride-sensitive Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule.

The effect of Ca2+, Cd2+, Ba2+, Mg2+ and pH on the renal epithelial Na(+)-channel was investigated by measuring the amiloride-sensitive 22Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule. It was found that intravesicular Ca2+ as well as extravesicular Ca2+ substantially lowered the channel-mediated flux. Amiloride sensitive Na+ uptake was nearly completely blocked by 10 microM Ca2+ at pH 7.4. The inhibitory effect of Ca2+ was dependent on pH. Thus, 10 microM Ca2+ produced 90% inhibition of 22Na+ uptake at pH 7.4, and only 40% inhibition at pH 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over the range from 7.0 to 7.4. All the cations Ca2+, Cd2+, Ba2+ except Mg2+ inhibited the 22Na+ influx drastically when added extravesicularly in millimolar concentrations. The cations Cd2+, Ba2+ and Mg2+ in the same concentrations intravesicularly inhibited the 22Na+ influx only slightly. A millimolar concentration of Ca2+ intravesicularly blocked the amiloride-sensitive 22Na+ flux completely. The data indicate that Ca2+ inhibits Na+ influx specifically by binding to sites composed of one or several deprotonated groups on the channel proteins.     

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).