Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes were studied by measuring 32P incorporation into di- and triphosphoinositide from Mg-[gamma-32P]ATP. The Km's for 32P incorporation into di- and triphosphoinositide were 110 and 48 microM ATP, respectively. The optimal temperature for 32P incorporation into diphosphoinositide was at 32 degrees C, whereas the optimum for triphosphoinositide labeling occurred at 43 degrees C. Differences in the effects of pH on the rate of 32P incorporation into di- and triphosphoinositide were also found. At 37 degrees C but not at 25 degrees C 32P-labeled diphosphoinositide was phosphorylated to triphosphoinositide in the presence of Mg-ATP. Triton X-100 partially inhibited 32P incorporation into diphosphoinositide but completely inhibited the synthesis of triphosphoinositide. At physiological concentrations, 0.4 mM MgCl2 half-maximally activated di- and triphosphoinositide synthesis. Higher concentrations of MgCl2 (5 to 50 mM) decreased 32P incorporation into diphosphoinositide and greatly enhanced 32P incorporation into triphosphoinositide. NaCl or KCl (less than or equal to 100 mM) did not have any effects on polyphosphoinositide synthesis, whereas 150 to 300 mM NaCl or KCl decreased synthesis of diphosphoinositide and increased synthesis of triphosphoinositide. Further studies showed that 50 mM MgCl2 and 200 mM NaCl or KCl stimulate kinase-mediated phosphorylation of diphosphoinositide to triphosphoinositide. Triton X-100 inhibited the ability of 50 mM MgCl2 and neomycin to stimulate phosphorylation of diphosphoinositide to triphosphoinositide. The pathways for synthesis of di- and triphosphoinositides in erythrocyte membranes are discussed.     

The H+-translocating ATP synthase in Halobacterium halobium differs from F0F1-ATPase/synthase

Cell envelope vesicles of Halobacterium halobium synthesize ATP by utilizing base-acid transition (an outside acidic pH jump) under optimal conditions (1 M NaCl, 80 mM MgCl2, pH 6.8) even in the presence of azide (a specific inhibitor of F0F1-ATPase) (Mukohata & Yoshida (1987) J. Biochem. 101, 311-318). An azide-insensitive ATPase was isolated from the inner face of the vesicle membrane, and shown to hydrolyze ATP under very specific conditions (1.5 M Na2SO4, 10 mM MnCl2, pH 5.8) (Nanba & Mukohata (1987) J. Biochem. 102, 591-598). This ATPase activity could also be detected when the vesicle components were solubilized by detergent. The relationship between ATP synthesis and the membrane-bound ATPase was investigated by modification of the vesicles with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) or N-ethylmaleimide (NEM). The inhibition pattern of ATP synthesis in the modified vesicles and that of ATP hydrolysis of the solubilized modified vesicles were compared under the individual optimum conditions. The inhibition patterns were almost identical, suggesting that the ATP synthesis and hydrolysis are catalyzed by a single enzyme complex. The ATP synthase includes the above ATPase (300-320 kDa), which is composed of two pairs of 86 and 64 kDa subunits. This is a novel H+-translocating ATP synthase functioning in the extremely halophilic archaebacterium. This "archae-ATP-synthase" differs from F0F1-ATPase/synthase, which had been thought to be ubiquitous among all respiring organisms on our biosphere.     

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.     

Regulation of ATP synthesis catalyzed by the calcium pump of sarcoplasmic reticulum

The role of pH, KCl, ATP, water activity, and temperature in ATP synthesis from ADP and Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In totally aqueous medium, the synthesis of ATP was inhibited by ATP, KCl, and pH values above 6.5. When the water activity of the medium was decreased by the addition of 30% (v/v) dimethyl sulfoxide, the synthesis of ATP was no longer inhibited by ATP; it was activated by KCl and the optimum pH changed from 6.5 to 7.5. In totally aqueous medium, the concentration of MgCl2 needed for half-maximal synthesis of ATP was found to vary with the temperature of the assay medium; at 35 degrees C it was 1 mM and increased to a value higher than 10 mM when the temperature was decreased to 15 degrees C. In the presence of 30% dimethyl sulfoxide, maximal synthesis of ATP was attained in presence of 0.05 mM MgCl2 at both 15 and 35 degrees C. The hypothesis is raised that in the living cell water structure may play a role in regulating the synthesis of ATP observed during the reversal of the Ca2+ pump of the sarcoplasmic reticulum.     

Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.     

Synthesis of ATP by soluble mitochondrial F1 ATPase and F1-inhibitor-protein complex in the presence of organic solvents

The F1 and F1-inhibitor-protein complex synthesized tightly bound ATP from ADP and Pi when the organic solvents dimethylsulfoxide (20-50% v/v), ethylene glycol (20-60% v/v) or poly(ethylene glycol) 4000 and 8000 (30-50% w/v) were included in the assay media. There was no synthesis of tightly bound ATP in the absence of organic solvents. In the presence of 50% dimethylsulfoxide, maximal synthesis of ATP was obtained at pH values between 6.5 and 7.7. In both F1 and F1-inhibitor-protein there was no synthesis of ATP in the absence of MgCl2. The rate of ATP synthesis became faster as the MgCl2 concentration in the medium was raised from 0.1-10 mM. The Km for Pi of F1 was in the range of 0.8-1.5 mM. The Km for Pi of the F1-inhibitor-protein was much higher than that of F1 and could not be measured. In the presence of 10 mM MgCl2 and 2 mM Pi, the rate constants of ATP synthesis by F1 and F1-inhibitor-protein were 5.2-10.4 h-1 and 3.5-5.9 h-1 respectively. For both enzymes the rate constant of ATP hydrolysis was 0.69 h-1. The tightly bound ATP of F1 and F1-inhibitor-protein were hydrolyzed at a much slower rate when either the Pi concentration or the MgCl2 concentration was suddenly decreased. Both in presence and absence of Mg2+, 40-60% of the radioactive tightly bound ATP synthesized by F1 was hydrolyzed when non-radioactive ATP was added to the assay medium. This was not observed when F1-inhibitor-protein was used.     

Purification and characterization of a membrane-associated ATPase from Natronococcus occultus, a haloalkaliphilic archaeon

Isolated membranes of the extreme haloalkaliphilic archaeon Natronococcus occultus were able to hydrolyze ATP via an ATPase, which required the presence of Mg(2+), high concentrations of NaCl, and a pH value of 9. The native molecular mass of the purified ATPase was 130 kDa and was composed of 74- and 61-kDa subunits. Enzyme activity was specific for the hydrolysis of ATP with slight activity towards GTP, CTP, and ITP. The enzyme required NaCl for maximal activity but Na(2)SO(4) and (NH(4))(2)SO(4) could substitute. The enzyme showed no activity if Na(2)SO(3) or sodium citrate was substituted for NaCl. The ATPase from N. occultus was inhibited by NBD-Cl, NaN(3), and ouabain, and was sensitive to nitrate, vanadate, DCCD, and bafilomycin A(1). It was not inhibited by NEM in contrast to other previously characterized halophile ATPases. The ATPase had a K(M) of 0.5 mM and appeared to be non-competitively inhibited by NaN(3) with a K(I) of 3.1 mM.     

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.     

Mitochondrial ATP synthase: dramatic Mg2+-induced alterations in the structure and function of the F1-ATPase moiety

The ATPase activity of the F1 moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 degrees C in the presence of MgCl2. The concentration of MgCl2 (130 microM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1 complex of alpha subunits and part of the gamma-subunit population. The latter form a precipitate while the beta, delta, and epsilon subunits, and the remaining part of the gamma-subunit population, remain soluble. Dissociation is not a sudden "all or none" event but parallels loss of ATPase activity until alpha subunits have almost completely dissociated together with about 50% of the gamma-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPi at high concentration (greater than or equal to 200 mM) is effective in partially protecting F1 against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0 moiety in F1-depleted particles. When bound to F0, F1 is protected completely against divalent cation induced inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and polymerization of skeletal muscle ADP-actin

Skeletal muscle ADP-G-actin was prepared from ADP-F-actin, which had been freed of residual ATP by repeated sonication, by depolymerization in 5 mM Tris-HCl, 0.2 mM ADP, 0.2 mM dithiothreitol, 0.1 mM CaCl2, 0.1 mM MgCl2, and 0.01% NaN3, pH 8.0. The ADP had been freed of traces of ATP by DEAE-chromatography, and 5 microM diadenosine pentaphosphate was added to inhibit myokinase activity. The kinetics of the spontaneous polymerization of ADP-actin in 1 mM MgCl2 + 0.1 M KCl were compatible with the simple nucleation-elongation model previously used to explain the polymerization of ATP-actin. The critical concentrations of ADP-actin were 8.0 and 2.0 microM in 1 mM MgCl2 and 1 mM MgCl2 + 0.1 M KCl, respectively. These values are 20-30-fold higher than the corresponding values in ATP. Using cross-linked actin trimers to nucleate polymerization, the association rate constants were found to be 0.8 and 0.9 microM-1 S-1 in MgCl2 and MgCl2 + KCl, respectively, which are 0.4 and 0.2 times the values for ATP-actin. The dissociation rate constants, calculated from the critical concentrations and the association rate constants, were 6.4 and 1.8 S-1, respectively, which are 10 and 5 times the corresponding values for ATP-actin.     

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.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

A membrane-bound ATPase from Halobacterium halobium: purification and characterization

An ATPase was newly identified on the inner face of the plasma membrane of the extremely halophilic archaebacterium Halobacterium halobium. The enzyme was released into an alkaline EDTA solution and purified by several chromatographic steps in the presence of sulfate at 1 M or over. The molecular weight of the native enzyme was around 320,000; it is most likely composed of two pairs (alpha 2 beta 2) of 86,000 (alpha) and 64,000 (beta) subunits. The enzyme hydrolyzed ATP and other nucleoside triphosphates but neither ADP nor AMP. The enzyme required divalent cations, among which Mn2+ was most effective (Mg2+ activated 35% of Mn2+). The ATPase activity was optimum at pH between 5.5 and 6, particularly in a nearly saturated Na2SO4 (or Na2SO3) solution, while it was very low in a chloride salt solution even at 4 M at any pH. The Km value for ATP was 1.4 mM and the K1 value for ADP (competitive to ATP) was 0.08 mM. Neither azide (a specific inhibitor for F0F1-and F1-ATPase) nor vanadate (for E1E2-ATPase) inhibited the enzyme. The ATPase was stable at high concentrations of sulfate. At low concentrations of salts, or at low temperatures even in high NaCl concentrations, the enzyme was inactivated. Although the ATPase isolated here from halobacterial membrane has such unusual characteristics, it is the most probable candidate for the (catalytic part of) halobacterial ATP synthase, which differs from F0F1-ATPase/synthase (Mukohata et al. (1986) J. Biochem. 99, 1-8; Mukohata and Yoshida (1987) J. Biochem. 101, 311-318).     

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.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.     

Characterization and function of catalytic subunit alpha of H+-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. A study with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Subunit alpha (Mr 89,000) from vacuolar membrane H+-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae was found to bind 8-azido[alpha-32P]adenosine triphosphate. Labeling by this photosensitive ATP derivative was saturable with an apparent dissociation constant of 10(-6) to 10(-5) M and decreased in the presence of ATP and ADP. The enzyme was inactivated by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), with about 1 microM causing half-maximal inactivation in the neutral pH range. This inactivation was prevented by the presence of ATP, ADP, or adenosyl-5'-yl imidodiphosphate (AMP-PNP). The original activity was restored by treating the inactivated enzyme with 2-mercaptoethanol. Kinetic and chemical studies of the inactivation showed that the activity was lost on chemical modification of a single tyrosine residue per molecule of the enzyme. When the enzyme was inactivated with [14C]NBD-Cl, subunit alpha was specifically labeled, and this labeling was completely prevented by the presence of ATP, GTP, ADP, or AMP-PNP. From these results, it was concluded that subunit alpha of yeast vacuolar H+-ATPase has a catalytic site that contains a single, essential tyrosine residue. The kinetics of single site hydrolysis of [gamma-32P]ATP (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100) indicated the formation of an enzyme-ATP complex and subsequent hydrolysis of bound ATP to ADP and Pi at the NBD-Cl-sensitive catalytic site. NBD-Cl inactivated the single site hydrolysis and inhibited the formation of an enzyme-ATP complex. Dicyclohexylcarbodiimide did not affect the single site hydrolysis, but inhibited the enzyme activity under steady-state conditions.     

Inhibition and labeling of the Ca2(+)-ATPase from sarcoplasmic reticulum by periodate oxidized ATP

The analog of ATP obtained by oxidation of the ribose ring of ATP with periodate (oxATP) was used as a reagent for the inhibition and labeling of the Ca2(+)-ATPase purified from sarcoplasmic reticulum membranes. The substrate concentration dependence for hydrolysis showed a biphasic pattern for both ATP and oxATP as substrates. Preincubation of Ca2(+)-ATPase in the presence of 0.05 mM CaCl2, 5 mM MgCl2, 100 mM KCl and oxATP led to an irreversible inhibition. This inhibition occurred faster at alkaline pH. The presence of ADP, adenyl-5'-imidodiphosphate (AMP-PNP) or EGTA in the preincubation medium decreased the rate of inhibition. OxATP covalently labels the enzyme: the labeling was decreased by ADP. This ADP-protected labeling increased with time until it reached approx. 1 mol [3H]oxATP per mol ATPase. The rate of labeling of the ADP-protected group correlated with the rate of loss of ADP-protected activity. Trypsin digestion of oxATP-labeled ATPase followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that fragment A1 contained a high degree of label that is displaced by ADP. We propose that the A1 fragment is situated close to the ribose ring when the adenosine moiety of ATP is bound to the catalytic site of the Ca2(+)-ATPase.     

Existence of ADP- and KCl-insensitive phosphoenzyme intermediate of Na+,K(+)-ATPase at alkaline Ph

The Na(+)-ATPase activity of Na+,K(+)-ATPase in the absence of K+ was least dependent on the sodium concentration when the pH was 9.5. Around 40% of the phosphoenzyme formed from ATP in the presence of 0.5 mM MgCl2 at alkaline pH was insensitive to both KCl and ADP. High-Na+ chase reversed this insensitivity, i.e., the phosphoenzyme became sensitive to KCl or ADP. On the other hand, phosphorylation at 0.1 mM MgCl2 instead of 0.5 mM showed at least 95% sensitivity to KCl. These observations suggest that ADP- and KCl-insensitive phosphoenzyme was formed when excess Mg++ was present during phosphorylation at alkaline pH. This phosphoenzyme might be an intermediate in the process of ATP hydrolysis.     

Imidazole chloride and tris-chloride substitute for sodium chloride in inducing high-affinity AdoPP[NH]P binding to (Na+ + K+)-ATPase

Optimal binding of [2,8-3H]AdoPP[NH]P to (Na+ + K+)-ATPase requires 25 mM Na+ (Cl-), 50 mM imidazole+ (Cl-) or 50 mM Tris+ (Cl-). Chloride is essential as counterion. We conclude that imidazole+ and Tris+ are able to bind to the Na+ site, and recommend the use of dilute buffers for studying the partial reactions of (Na+ + K+)-ATPase. In NaCl or the substituting buffers the dissociation constant for the enzyme-AdoPP[NH]P complex at 0 degrees C and pH 7.25 is 0.4 microM, whereas in millimolar MgCl2 it is about 2 microM. These distinct levels in affinity with MgCl2 as compared to NaCl, together with the MgCl2-dependence of photolabelling of the enzyme with ATP analogues (Rempeters, G. and Schoner, W. (1981) Eur. J. Biochem. 121, 131-137), suggest significant changes within the substrate site of (Na+ + K+)-ATPase upon binding of Mg2+ (Cl-)2.     

Interaction of ATP with sarcoplasmic reticulum Ca2+-ATPase; effect on the conformational state of the enzyme

It was studied how temperature influences the NBD-Cl inactivation of sarcoplasmic reticulum Ca2+-ATPase and the protective effect of ATP under conditions preventing ATP hydrolysis. Two types of ATP-binding sites with Kd equal to 30 and 220 microM at 37 degrees C were found. ADP interacts with these sites with the (K'd = 20 and 200 microM). The temperature decrease from 25 degrees to 5 degrees C induces the abrupt increase in the Kd for the low affinity site. The possible reasons for heterogeneity of ATP-binding sites are discussed. The conclusion is made that interaction of monomers in oligomeric complex of Ca2+-ATPase induces heterogeneity of ATP-binding sites.