Steady-state Level Of Phosphoenzyme Research Articles

Inhibition of plasma membrane Ca2+-ATPase by heparin is modulated by potassium

Heparin is related to several protein receptors that control Ca2+ homeostasis. Here, we studied the effects of heparin on the plasma membrane Ca2+-ATPase from erythrocytes. Both ATP hydrolysis and Ca2+ uptake were inhibited by heparin without modification of the steady-state level of phosphoenzyme formed by ATP. Calmodulin did neither modify the inhibition nor the binding of heparin. Inhibition by heparin was counteracted by K+ but not by Li+. This effect was extended to other sulfated polysaccharides with high number of sulfate residues. Hydrolysis of p-nitrophenylphosphate was equally inhibited by heparin. No evidence for enzyme uncoupling was observed: Ca2+ uptake and ATP hydrolysis remained tightly associated at any level of heparin, and heparin did not increase the passive Ca2+ efflux of inside-out vesicles. Vanadate blocked this efflux, indicating that the main point of Ca2+ escape from these vesicles was linked to the Ca2+ pump. It is discussed that sulfated polysaccharides may physiologically increase the steady-state level of Ca2+ in the cytosol by inhibiting the Ca2+ pumps in a K+ (and tissue) regulated way. It is suggested that heparin regulates the plasma membrane Ca2+-ATPase by binding to the E2 conformer.

Curcumin modulation of Na,K‐ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity

1 Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca(2+)-ATPase. 2 We tested the hypothesis that curcumin may modulate other members of the P(2)-type ATPase superfamily by studying the effects of curcumin on the activity and kinetic properties of the Na,K-ATPase. 3 Curcumin treatment inhibited Na,K-ATPase activity in a dose-dependent manner (K(0.5) approximately 14.6 microM). Curcumin decreased the apparent affinity of Na,K-ATPase for K(+) and increased it for Na(+) and ATP. Kinetic analyses indicated that curcumin induces a three-fold reduction in the rate of E1P --> E2P transition, thereby increasing the steady-state phosphoenzyme level. Curcumin treatment significantly abrogated K(+) occlusion to the enzyme as evidenced from kinetic and proteolytic cleavage experiments. Curcumin also significantly decreased the vanadate sensitivity of the enzyme. 4 Thus, curcumin partially blocks the K(+) occlusion site, and induces a constitutive shift in the conformational equilibrium of the enzyme, towards the E1 conformation. 5 The physiological consequences of curcumin treatment previously reported in different epithelial model systems may, at least in part, be related to the direct effects of curcumin on Na,K-ATPase activity.

Functional effect of hydrogen peroxide on the sarcoplasmic reticulum membrane: uncoupling and irreversible inhibition of the Ca 2+-ATPase protein

The chemical treatment of sarcoplasmic reticulum vesicles with H 2O 2 affects both Ca 2+ transport and the hydrolytic activity supported by the Ca 2+-ATPase protein. Ca 2+ transport was much more sensitive to inhibition than ATPase activity and the decrease in Ca 2+ transport was not the result of an increase in membrane permeability. The Ca 2+/P i uncoupling can be attributed to the own catalytic mechanism of the enzyme. Under conditions of high uncoupling, Ca 2+ binding to the transport sites was barely affected and accumulation of phosphorylated species during the enzyme cycling gave almost maximal levels. These are features defining intramolecular uncoupling mediated by a phosphorylated form of the enzyme. Severe inhibition of the hydrolytic activity was observed when higher peroxide concentrations and leaky vesicles were used. These experimental conditions diminished maximal Ca 2+ binding and the steady-state phosphoenzyme level. The low hydrolytic activity can be ascribed to a decrease in the rate of enzyme dephosphorylation.

Kinetics Studies of the Cardiac Ca-ATPase Expressed in Sf21 Cells: New Insights on Ca-ATPase Regulation by Phospholamban

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells ( Spodoptera frugiperda insect cells) have been carried out to test the hypotheses that phospholamban inhibits Ca-ATPase cycling by decreasing the rate of the E1·Ca to E1′·Ca transition and/or the rate of phosphoenzyme hydrolysis. Three sample types were studied: Ca-ATPase expressed alone, Ca-ATPase coexpressed with wild-type phospholamban (the natural pentameric inhibitor), and Ca-ATPase coexpressed with the L37A-phospholamban mutant (a more potent monomeric inhibitor, in which Leu 37 is replaced by Ala). Phospholamban coupling to the Ca-ATPase was controlled using a monoclonal antibody against phospholamban. Gel electrophoresis and immunoblotting confirmed an equivalent ratio of Ca-ATPase and phospholamban in each sample (1 mol Ca-ATPase to 1.5 mol phospholamban). Steady-state ATPase activity assays at 37°C, using 5 mM MgATP, showed that the phospholamban-containing samples had nearly equivalent maximum activity (∼0.75 μmol·nmol Ca-ATPase −1·min −1 at 15 μM Ca 2+), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K Ca values by 200 nM and 400 nM, respectively. When steady-state Ca-ATPase phosphoenzyme levels were measured at 0°C, using 1 μM MgATP, the K Ca values also shifted by 200 nM and 400 nM, respectively, similar to the results obtained by measuring ATP hydrolysis at 37°C. Measurements of the time course of phosphoenzyme formation at 0°C, using 1 μM MgATP and 268 nM ionized [Ca 2+], indicated that L37A-phospholamban decreased the steady-state phosphoenzyme level to a greater extent (45%) than did wild-type phospholamban (33%), but neither wild-type nor L37A-phospholamban had any effect on the apparent rate of phosphoenzyme formation relative to that of Ca-ATPase expressed alone. Measurements of inorganic phosphate (P i) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P i release to a greater extent (45%) than did wild-type phospholamban (33%). However, independent measurements of Ca-ATPase dephosphorylation after the addition of 5 mM EGTA to the phosphorylated enzyme showed that neither wild-type phospholamban nor L37A-phospholamban had any effect on the rate of phosphoenzyme decay relative to Ca-ATPase expressed alone. Computer simulation of the kinetics data indicated that phospholamban and L37A-phospholamban decreased twofold and fourfold, respectively, the equilibrium binding of the first Ca 2+ ion to the Ca-ATPase E1 intermediate, rather than inhibiting rate of the E·Ca to E′·Ca transition or the rate of phosphoenzyme decay. Therefore, we conclude that phospholamban inhibits Ca-ATPase cycling by decreasing Ca-ATPase Ca 2+ binding to the E1 intermediate.

Leucine 332 at the boundary between the fourth transmembrane segment and the cytoplasmic domain of Na+,K+-ATPase plays a pivotal role in the ion translocating conformational changes.

Mutants Gly330-->Ala, Leu332-->Ala, Leu332-->Pro, and Pro780-->Ala of the alpha1-isoform of rat kidney Na+,K+-ATPase were expressed in COS-1 cells to active site concentrations between 20 and 70 pmol per mg of membrane protein. The functional properties of the mutants were characterized by titrations of Na+-, K+-, and ATP-dependencies of Na+,K+-ATPase activity as well as by a series of assays measuring the K+-dependence of the steady-state phosphoenzyme level, the kinetics of dephosphorylation under a variety of conditions, and the ADP-ATP exchange activity. In mutants Gly330-->Ala, Leu332-->Ala, and Leu332-->Pro, the molecular turnover number was reduced relative to that of the wild-type Na+,K+-ATPase, and the steady-state phosphoenzyme level was high even in the presence of several millimolar K+. At a low Na+ concentration in the absence of K+, mutants Leu332-->Pro and Gly330-->Ala displayed high ADP-ATP exchange activity and formed a high level of the ADP-sensitive phosphoenzyme (E1P). The phosphoenzyme decayed slowly following a jump in salt concentration and chase with ATP and K+. Hence, the conversion of E1P to the K+-sensitive phosphoenzyme (E2P) was inhibited in mutants Leu332-->Pro and Gly330-->Ala. In the Leu332-->Ala mutant, a high level of E2P was accumulated in the absence of K+, and the ADP-ATP exchange activity was low at low Na+ concentration in the absence of K+, but rose sharply on addition of K+. Dephosphorylation experiments indicated that in the Leu332-->Ala mutant K+ induced reversal of the phosphoenzyme interconversion, forming E1P from E2P. Leu332 is therefore a pivotal residue in the conformational change. Mutants Gly330-->Ala and Pro780-->Ala displayed reduced K+ affinities relative to the wild type, determined in three independent assays.

Pre-steady-state kinetic study of the effects of K+ on the partial reactions of the catalytic cycle of the plasma membrane Ca2+-ATPase

The effects of 100 mM K+ on the partial reactions that take place during ATP hydrolysis on the calcium ion-dependent ATPase from plasma membrane (PM-Ca(2+)-ATPase) were studied at 37 degrees C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. At 10 microM [gamma-32P]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the k(app) of formation by 140% to 84 11 s-1 and the steady-state level of phosphoenzyme (EP) by 25% to 3.4 0.17 pmol/mg of protein. If added together with [gamma-32P]ATP at the beginning of phosphorylation, K+ was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the E2 --> E1 transition by phosphorylation showed that in medium with Ca2+ and Mg2+, K+ increased the k(app) of the transition by 55% to 14 3 s-1 and the apparent concentration of E1 by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with K+. The presence of K+ did not change the slow decay of EP without Mg2+ but activated the decay of EP made with Mg2+, increasing its k(app) by 60% to 91 12 s-1. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation K+ was fully effective in favouring decomposition of EP made in medium containing no K+. In the presence of either 3mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaE2P, the effect of K+ on dephosphorylation was conserved. Because the sites for K+ are intracellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca(2+)-ATPase under physiological conditions.

Functional consequences of mutation Asn 326→Leu in the 4th transmembrane segment of the a-subunit of the rat kidney Na +,K +-ATPase

Site-specific mutagenesis was used to replace Asn 326 in transmembrane segment M4 of the ouabain-insensitive α 1-isoform of rat kidney Na +,K +-ATPase. Mutant Asn 326 → Leu was functional as demonstrated by the ability of COS cells expressing the mutant enzyme to grow in the presence of ouabain. In three independent assays encompassing Na + titrations of Na +,K +-ATPase activity, Na +-ATPase activity, and phosphorylation from ATP, the Asn 326 → Leu mutant displayed a reduced apparent affinity for Na +. By contrast, this mutant exhibited a slightly increased apparent affinity for K + relative to the wild-type enzyme. In the presence of Na +. without K +, the Asn 326 → Len mutant hydrolyzed ATP at a high rate corresponding to 32% of the maximal Na +,K +-ATPase activity, and the rate of dephosphorylation of the phosphoenzyme intermediate was enhanced in the mutant relative to that of the wild-type enzyme. Oligomycin, known to stabilize the Na +-occluded phosphoenzyme intermediate, reduced the dephosphorylation rate of the mutant and increased the steady-state phosphoenzyme level formed by the mutant at least 3-fold, whereas an increase in the steady-state phosphoenzyme level of only 10–15% was determined for the wild-type enzyme. The molecular turnover number for the Na +,K +-ATPase reaction, calculated when the steady-state phosphoenzyme level obtained in the presence of oligomycin was taken as a measure of the concentration of active sites, was slightly reduced relative to that of the wild-type enzyme. The data are discussed in terms of a role for Asn326 in binding of cytoplasmic Na + and in mediation of inhibition of dephosphorylation.

Effects of magnesium plus vanadate on partial reactions of the Ca 2+-ATPase from human red cell membranes

Under conditions in which pretreatment with Mg 2+ plus vanadate activate the Ca 2+-ATPase, the initial rate of phosphorylation of the enzyme increased from 141 to 259 pmol/mg protein per s while the steady-state level of phosphoenzyme lowered from 1.9 to 1.1 pmol/mg protein. The drop in phosphoenzyme level was caused by incubation and washing during treatment rather than by vanadate. The data allowed to estimate a turnover number for the enzyme that raised by 170% after pretreatment. The results show that the activation of the Ca 2+-ATPase by Mg 2+ plus vanadate is due to changes in the kinetic properties of the enzyme.

Mutant Glu781-->Ala of the rat kidney Na+,K(+)-ATPase displays low cation affinity and catalyzes ATP hydrolysis at a high rate in the absence of potassium ions.

Site-specific mutagenesis was used to replace Glu329, Glu781, Asp806, Thr809, and Asp810 in the transmembrane domain of the ouabain-insensitive alpha 1-isoform of rat kidney Na+,K(+)-ATPase. cDNAs encoding any of the mutants Glu329-->Ala, Glu781-->Ala, Asp806-->Asn, Thr809-->Ala, and Asp810-->Asn were transfected into COS cells, and transfectants were grown in the presence of ouabain to inhibit the endogenous COS cell Na+,K(+)-ATPase. Mutants Glu781-->Ala and Thr809-->Ala were functional as evidenced by their ability to confer ouabain resistance to the cells, whereas mutants Glu329-->Ala, Asp806-->Asn, and Asp810-->Asn were inactive. The apparent Na+ affinities determined by titrations of Na+,K(+)-ATPase activity, Na(+)-ATPase activity, and phosphorylation from ATP in mutants Glu781-->Ala and Thr809-->Ala were strongly reduced relative to the affinity of the wild type (6-8-fold increase in K0.5 for Na+ in the phosphorylation assay for both mutants). The Glu781-->Ala mutant displayed a 3-4-fold reduction in the apparent affinity for K+ and was able to hydrolyze ATP at a high rate in the absence of K+ (Vmax for Na(+)-ATPase activity 5-fold higher than that of the wild-type enzyme). The steady-state phosphoenzyme level formed by the Glu781-->Ala mutant was increased 3-fold by addition of oligomycin, whereas only a slight effect of oligomycin was observed for mutant Thr809-->Ala and the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of phosphoenzyme formation from phosphate and sarcoplasmic reticulum Ca(2+)-ATPase by vanadate binding to high- or low-affinity site on the enzyme.

In the preceding paper, we suggested that 1 mol Ca(2+)-ATPase of sarcoplasmic reticulum (SR) contains 0.5 ml of high-affinity vanadate binding sites as well as 0.5 ml of low-affinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (Pi). When vanadate was added to the reaction medium in which the Ca(2+)-ATPase had been phosphorylated by Pi in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17 microM, and reached maximum at 50 microM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of E2P was competitively prevented by Pi with a dissociation constant of 7.4 mM, which was very close to Km for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with Pi at a phosphorylation site on the Ca(2+)-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca(2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

A K(+)-competitive fluorescent inhibitor of the H,K-ATPase

The interactions of a novel fluorescent compound, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ) with the gastric H,K-ATPase were determined. MDPQ was shown to inhibit the H,K-ATPase and its associated K(+)-phosphatase competitively with K+, with Ki values of 0.22 and 0.65 microM, respectively. It also inhibited H+ transport with an IC50 of 0.29 microM, but at a concentration of 3.5 microM, reduced the steady-state level of phosphoenzyme by only 28%. The fluorescence of the inhibitor increased upon binding to the enzyme. 70% of this increment was quenched by K+, independently of Mg2+. The binding of MgATP to a high affinity site (K0.5(ATP) less than 1 microM) markedly increased the fluorescence due to the formation of an inhibitor-phosphoenzyme complex saturating with a K0.5(MDPQ) of 0.94 microM. The K(+)-dependent fluorescent quench (K0.5(K+) = 1.8 mM) required the ionophore, nigericin, indicating that K+ and MDPQ were competing at an extracytosolic site on the enzyme. Formation also of an enzyme-vanadyl-inhibitor complex was shown by the fact that Mg2+ plus vanadate enhanced MDPQ fluorescence in the absence of MgATP and decreased fluorescence in the presence of MgATP. The minimal stoichiometry of bound MDPQ determined by fluorescence titrations in the presence of MgATP was 1.4 mol/mol phosphoenzyme. The data suggest that this compound can serve as a probe of conformation at an extracytosolic site of the H,K-ATPase.

Interaction of a K(+)-competitive inhibitor, a substituted imidazo[1,2a] pyridine, with the phospho- and dephosphoenzyme forms of H+, K(+)-ATPase.

Defining the structural and catalytic properties of the ion transport site(s) of enzyme-phosphorylating ATPases is of key importance in understanding the mechanism of ion transport by these enzymes. In the case of the H+, K(+)-ATPase, SCH 28080 (3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a]-pyridine) has been shown to act as a high affinity, extracytosolic, K(+)-competitive inhibitor of Mg2+, K(+)-ATPase activity (Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson, R., Mendlein, J., Rabon, E., and Sachs, G. (1987) J. Biol. Chem. 262, 2077-2084). To define the nature of the SCH 28080-binding site in relation to the catalytic cycle of the enzyme, we have investigated the effects of this potential K+ transport site probe on the steady-state and partial reactions of the H+, K(+)-ATPase. In the absence of K+, SCH 28080 inhibits Mg2(+)-ATPase activity with high affinity (apparent Ki = 30 nM). Inhibition is due to K(+)-like prevention of phosphoenzyme formation. SCH 28080 has no effect on Mg2(+)-catalyzed dephosphorylation. SCH 28080, at concentrations less than 0.5 microM, increases the apparent Km for K+ for Mg2+, K(+)-ATPase activity with little effect on the maximum velocity. At higher concentrations of SCH 28080, reversal of inhibition by higher K+ concentrations is not complete, due to inhibition of ATPase activity by high K+. In contrast, SCH 28080 inhibits K(+)-stimulated dephosphorylation by competitively displacing K+ from phosphoenzyme with an extracytosolic conformation of the monovalent cation site (E2P) at low concentrations of SCH 28080 and K+. At higher concentrations, 10 microM SCH 28080 and 50 mM K+, a slowly dephosphorylating complex with both SCH 28080 and K+ bound to E2P may form which represents a small fraction of the total E2P (15-25%). Preincubation of SCH 28080 with E2P completely blocks K(+)-stimulated dephosphorylation, and K+ is unable to reverse this preincubation effect, indicating that the SCH 28080 dissociation rate is at least as slow as K(+)-independent dephosphorylation of E2P. These findings indicate that SCH 28080 inhibits K(+)-stimulated ATPase activity by competing with K+ for binding to E2P and blocking K(+)-stimulated dephosphorylation. In the absence of K+, SCH 28080 has a higher apparent affinity for E2P, but it permits K(+)-independent dephosphorylation. Since the dissociation rate of SCH 28080 from the enzyme is slow, phosphoenzyme formation is prevented by SCH 28080 remaining bound to the extracytosolic conformation of the monovalent cation site, thereby reducing the steady-state level of phosphoenzyme.

Effect of ADP on the rate of acetyl phosphate hydrolysis by the Ca2+‐ATPase of sarcoplasmic reticulum

Hydrolysis of acetyl phosphate is inhibited by high concentrations of Pi and MgCl2, probably due to an increase in the steady-state level of phosphoenzyme formed from Pi in the medium. A dual effect of ADP during steady-state hydrolysis of acetyl phosphate was observed. ADP inhibited hydrolysis in the presence of 5 mM MgCl2 and no added Pi, whereas it stimulated hydrolysis when phosphoenzyme formation by Pi was favored by including 6 mM Pi and 20 mM MgCl2 in the assay medium. ATP inhibited acetyl phosphate hydrolysis in both of these assay media. When phosphoenzyme formation by Pi in the presence of acetyl phosphate was stimulated at Ca2+ concentrations sufficient to saturate the low-affinity Ca2+-binding sites, ADP stimulated acetyl phosphate hydrolysis and also promoted ATP synthesis by reversal of the catalytic cycle. The rate of ATP synthesis was dependent on ADP, Pi and Ca2+. Phosphoenzyme formation by Pi and MgCl2, whether in the absence of Ca2+ and acetyl phosphate, or during acetyl phosphate hydrolysis, was inhibited by ADP and ATP. These results suggest that ADP interacts with different intermediates of the catalytic cycle and that expression of inhibition or activation of acetyl phosphate hydrolysis depends on the steady-state level of phosphoenzyme formed by Pi.

Kinetic studies of the gastric H,K-ATPase. Evidence for simultaneous binding of ATP and inorganic phosphate.

The steady state rate of ATP hydrolysis (v) by the gastric H,K-ATPase and the steady state level of phosphoenzyme (E-P) have been measured at 0 and 10 mM KCl; both v and E-P have a nonhyperbolic dependence on the ATP concentration that is consistent with negative cooperativity. The ratio of the rate of hydrolysis to phosphoenzyme (v/[E-P]) was found to vary with the concentration of ATP. Thus, for the rate law v = [E-P].k, k must be a function of the ATP concentration. This requires that ATP be able to bind to E-P or to an enzyme form that occurs after E-P but prior to an irreversible step, such as the loss of inorganic phosphate (Pi). At low ATP concentrations, product inhibition by Pi gives concave downward plots of 1/v against Pi concentration. Pi increases the apparent Km and decreases the apparent Vm. At saturating ATP concentrations, Pi is a noncompetitive inhibitor. These data show that ATP and Pi can bind to the H,K-ATPase simultaneously. They are inconsistent with mechanisms where the binding of ATP and Pi is mutually exclusive.

Pre-steady-state phosphorylation of the human red cell Ca2+-ATPase.

The pre-steady-state kinetics of phosphorylation of the Ca2+-ATPase by ATP was studied at 37 degrees C and in intact red cell membranes to approach physiological conditions. ATP and Ca2+ activate with K0.5 of 4.9 and 26.4 microM, respectively. Preincubation with Ca2+ did not change the K0.5 for ATP. Preincubation with ATP did not alter the initial velocity of phosphorylation suggesting that binding of ATP was not rate-limiting. Mg2+ added at the start of the reaction increased the initial rate of phosphorylation from 4 to 8 pmol/mg/s. With 30 microM Ca2+, the K0.5 for Mg2+ was 60 microM. Mg2+ and Ca2+ added together beforehand accelerated phosphorylation to 70 pmol/mg/s. Phosphorylation of calmodulin-bound membranes was the fastest (280 pmol/mg/s), and its time course showed a neat overshoot before steady state. The results suggest that either preincubation with Ca2+ plus Mg2+ or calmodulin accelerated phosphorylation shifting toward E1 the equilibrium between the E1 and E2 conformers of the enzyme. K+ had no effect on the initial rate of phosphorylation and lowered by 40% the steady-state level of phosphoenzyme in the absence of Mg2+. Phosphorylation is not rate-limiting for the overall reaction since its initial rate was always higher than ATPase activity. In the absence of K+, the turnover of the phosphoenzyme was 2000 min-1, which is close to the values for other transport ATPases.

Protons as substitutes for sodium and potassium in the sodium pump reaction.

The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.

Inhibitory effects of cations on the gastric H+, K+ -ATPase. A potential-sensitive step in the K+ limb of the pump cycle.

The presence of a cation inhibitory site on the dephosphoform of the H+, K+ -ATPase was confirmed by comparing the effects of K+ and NH4+ on overall activity and on phosphorylation and dephosphorylation. Inhibition of ATPase activity was pronounced at high cation/ATP ratios, but NH4+ was much less effective. At 60 mM cation, although the ATPase activity was greater in the presence of NH4+ (17.1 mumol/mg.h) as compared to K+ (5.1 mumol/mg.h), dephosphorylation of preformed phosphoenzyme was faster with K+ (2101 min-1) than with NH4+ (1401 min-1). Increasing K+ concentrations at the cytosolic face of the enzyme, at constant ATP, decreased the rate of phosphorylation from 1343 to 360 min-1 at 25 mM K+. Increasing ATP concentrations in the presence of constant K+ concentrations accelerated ATPase activity and increased the steady-state phosphoenzyme level. Therefore, inhibition by cations was due to cation stabilization of a dephospho form of the enzyme at a cytosolically accessible cation-binding site. ATP promoted cation dissociation from this site. In ion-permeable vesicles, increasing K+ concentrations, at constant ATP, activated and then inhibited ATPase activity, with a K0.5(I) of 22 mM. In intact, ion-impermeable inside-out vesicles, in the presence of valinomycin, ATPase activity increased up to 175 mM K+. Collapse of this potential by the addition of the electrogenic protonophore 3,3',4', 5-tetrachlorosalicylanilide restored the K+ inhibition of ATPase activity. Thus, the cation inhibition of the ATPase activity appears to be voltage-sensitive; and hence, its connection to the voltage sensitivity of acid secretion demonstrated in intact gastric mucosa is discussed.

Tryptic cleavage inhibits but does not uncouple Ca2+ATPase of sarcoplasmic reticulum

Sarcoplasmic reticulum Ca2+ATPase is cleaved by trypsin at two sites, T1 and T2. Cleavage at T1 is complete, whereas only about 50% of the Ca2+ATPase is digested at the T2 site. In the absence of Ca2+ ionophor, Ca2+-ATPase activity of the digested enzyme remains virtually unchanged. In the presence of Ca2+ ionophor, however, the calculated specific activity of the doubly cleaved Ca2+ATPase is decreased by about 40%. The decrease in Ca2+ transport activity is much more rapid than cleavage of the T2 site, and could be correlated with an increased leak of Ca2+ from the digested vesicles. We obtained evidence that this leakiness is independent of the digestion of the Ca2+ATPase itself and is presumably due to the digestion of some other components of the sarcoplasmic reticulum vesicles. Examination of steady-state phosphoenzyme levels resulting from phosphorylation by ATP and Pi, or dephosphorylation by ADP or ADP/EGTA revealed no difference between the digested and the undigested Ca2+ATPase indicating no change in the equilibria caused by the T2 cleavage. Analysis of the substrate concentration dependence of the Ca2+ATPase activity also led to the conclusion that the digestion at T2 reduced the Vmax of ATP hydrolysis but leaves the Km unchanged. The above results are consistent with the model that cleavage at the T2 site reduces the turnover rate of the Ca2+ATPase reaction cycle by about 40% by slowing down or altering the rate-limiting step without affecting the equilibrium constants of the examined steps. We found no evidence of true uncoupling of Ca2+ transport from ATP hydrolysis correlated with cleavage at the T2 site.

A spin probe study of the water associated with a steady-state level of phosphoenzyme of the Ca-ATPase

The electron spin resonance spectrum of a water-soluble nitroxide free radical TEMPOL has been measured in concentrated suspensions of sarcoplasmic reticulum vesicles. Spectra observed in the presence of a high steady-state concentration of phosphoenzyme, formed from inorganic phosphate in the presence of Mg 2+, have been compared with those observed in the absence of phosphoenzyme. Quite large and reproducible differences in the two spectra were observed. Below 0 ° C the bulk water froze, and TEMPOL accumulated into a residual liquid phase of high microscopic viscosity, and of polarity slightly less than water. When lipids were extracted from the vesicles, dispersed in water and frozen, the residual signal from TEMPOL showed that it was in an environment of polarity similar to ethanol. It is proposed that the water which did not freeze at 0 ° C was contained in the hydrophobic cleft of the ATPase containing the active site. Like water in small hydrophobic pores of synthetic polymers, it consisted of large strongly-bonded clusters, and had high viscosity, low entropy and profoundly changed solvent properties.

Papain fragmentation of the gastric (H + + K +)-ATPase

Membrane-bound (H + + K +)-ATPase purified from hog gastric mucosa was exposed to limited papain digestion. Such treatment resulted in a rapid inhibition of the K +-stimulated adenosine triphosphatase and p-nitrophenyl phosphatase activities, with about 90% of these activities lost after 3 min incubation at 37°C with 0.1 units of papain per mg of enzyme protein. Parallel to the inhibition of the enzyme activities, there was a production of a 77 kDa membrane-bound fragment containing the aspartyl phosphate residue of the phospho-intermediate. This fragment accounted for about 45% of the total enzyme protein after the 3 min papain treatment. The digestion barely affected the steady-state level of phosphorylation, allowed the aspartyl phosphate of the 77 kDa fragment to undergo the transition to the E 2P form, and did not significantly alter the fraction of ADP-sensitive phosphoenzyme. The presence of KCl, however, depressed the steady-state level of phosphoenzyme formed from [γ- 32P]ATP considerably less than that of the control enzyme. With further exposure to papain the 77 kDa peptide became fragmented into a 28 kDa soluble peptide that retained the phosphorylating site. Binding of fluorescein 5′-isothiocyanate (FITC) to the native enzyme did not affect the sites of papain hydrolysis because the same peptide fragments were obtained. The FITC reaction site was also in the 28 kDa soluble peptide fragment.