Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

Changes in affinity for calcium ions with the formation of two kinds of phosphoenzyme in the Ca2+,Mg2+-dependent ATPase of sarcoplasmic reticulum

The amount of Ca2+ bound to the Ca2+,Mg2+-dependent ATPase of deoxycholic acid-treated sarcoplasmic reticulum was measured during ATP hydrolysis by the double-membrane filtration method [Yamaguchi, M. & Tonomura, Y. (1979), J. Biochem. 86, 509--523]. The maximal amount of phosphorylated intermediate (EP) was adopted as the amount of active site of the ATPase. In the absence of ATP, 2 mol of Ca2+ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca2+ binding to the ATPase in the presence of MgCl2. Under the conditions where most EP and ADP sensitive at steady state (58 microM Ca2+, 50 microM EGTA, and 20 mM MgCl2 at pH 7.0 and 0 degrees C), bound Ca2+ increased by 0.6--0.7 mol per mol active site upon addition of ATP. The time course of decrease in the amount of bound 45Ca2+ on addition of unlabeled Ca2+ + EGTA was biphasic, and 70% of bound 45Ca2+ was slowly displaced with a rate constant similar to that of EP decomposition. Similar results were obtained for the enzyme treated with N-ethylmaleimide, which inhibits the step of conversion of ADP-sensitive EP to the ADP-insensitive one. Under the conditions where most EP was ADP insensitive at steady state (58 microM Ca2+, 30 microM EGTA, and 20 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ increased slightly, then decreased slowly by 1 mol per mol of EP formed after addition of ATP. Under the conditions where about a half of EP was ADP sensitive (58 microM Ca2+, 25 microM EGTA, and 1 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ did not change upon addition of ATP. These findings suggest that the Ca2+ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.     

Chemical modification of an arginine residue in the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by phenylglyoxal

Phenylglyoxal (PGO) was used as a reagent for chemical modification of the ATP-binding site of Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum (SR-ATPase). When 1 mM PGO was reacted with SR-ATPase at 30°C at pH 8.5, PGO was bound to the ATPase molecule in two-to-one stoichiometry with concomitant loss of activity of the ATPase to form the phosphorylated intermediate (E-P). ATP and ADP prevented the binding of PGO and thereby protected the enzyme from inactivation. The SR membranes were labeled with [14C]PGO and then digested with pepsin to identify the attachment site of PGO. A 14C-labeled peptide (402lle-Arg*-Ser-Gly-Gln406) was purified to homogeneity by C18-reversed phase HPLC (Arg* denotes the binding site of [14C]PGO). These results indicate that Arg403 is located in the ATP binding site of the SR-ATPase.     

Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.     

Reaction of a carbodiimide adduct of ATP at the active site of sarcoplasmic reticulum calcium ATPase

An adduct of a carbodiimide and ATP was synthesized from 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and the nucleotide. Despite its limited stability (t1/2 for hydrolysis of about 5 min at 25 degrees C), it was shown to react with and inactivate the calcium ATPase of sarcoplasmic reticulum in its vesicular, nonionic detergent-solubilized and purified forms. Saturation kinetics, with an ATP-EDC concentration dependence midpoint in the 10 microM range, were observed, suggesting an active-site affinity which is similar to ATP. The reaction was specific in that inactivation required reaction of about one adduct per ATPase. The modified enzyme could no longer be phosphorylated by ATP or Pi or hydrolyze p-nitrophenyl phosphate, but retained the ability to undergo the high-affinity calcium-dependent fluorescence change. It also bound trinitrophenyl-ADP and other nucleotides at least 10-fold more weakly than the unmodified ATPase. The inactivation reaction required the presence of Mg2+ and Ca2+ and was prevented by nucleotides such as ATP and ADP. For magnesium, the inactivation-enabling effect occurred with a midpoint of 3 mM. In the case of calcium, the transition resembled high-affinity binding in that it occurred cooperatively with a midpoint in the micromolar range. Higher [Mg2+] shifted this transition to higher [Ca2+]. Polyacrylamide gel electrophoresis (PAGE) demonstrated that the reaction converted the ATPase (Mr = 1.1 x 10(5)) to a species with an apparent Mr = (1.7-1.8) x 10(5). Since nonionic detergent-solubilized ATPase and purified ATPase gave similar results, intramolecular cross-linking is implicated.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H-NMR studies on nucleotide binding to the sarcoplasmic reticulum Ca2+ ATPase. Determination of the conformations of bound nucleotides by the measurement of proton-proton transferred nuclear Overhauser enhancements

The glycosidic bond torsion angles and the conformations of the ribose of Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P (magnesium adenosine 5'-[beta, gamma-imido]triphosphate) bound to Ca2+ATPase, both native and modified with fluorescein isothiocyanate (FITC), in intact sarcoplasmic reticulum have been determined by the measurement of proton-proton transferred nuclear Overhauser enhancements by 1H-NMR spectroscopy. This method shows clearly the existence of a low-affinity ATP binding site after modification of the high-affinity site with FITC. For all three nucleotides bound to both the high-affinity (catalytic) site and the low-affinity site, we find that the conformation about the glycosidic bond is anti, the conformation of the ribose 3'-endo of the N type and the conformation about the ribose C4'-C5' bond either gauche-trans or trans-gauche. The values for the glycosidic bond torsion angles chi (O4'-C1'-N9-C4) for Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P bound to the low-affinity site of FITC-modified Ca2+ATPase are approximately equal to 270 degrees, approximately equal to 260 degrees and approximately equal to 240 degrees respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ATPase, chi lies in the range 240-280 degrees.     

Simulation of cardiac work transitions, in vitro: effects of simultaneous Ca2+ and ATPase additions on isolated porcine heart mitochondria

During increases in cardiac work there are net increases in cytosolic [Ca(2+)] and ATP hydrolysis by myofiliments and ion transport ATPases. However, it is still unclear what role Ca(2+)or the ATP hydrolysis products, ADP and Pi, have on the regulation of mitochondrial ATP production. In this study, work jumps were simulated by simultaneous additions of Ca(2+) and ATPase to porcine heart mitochondria. The net effects on the mitochondrial ATP production were monitored by simultaneously monitoring respiration (mVo2), [NADH], [ADP] and membrane potential (deltapsi) at 37 degrees C. Addition of exogenous ATPase (300 mlU.ml(-1))]ATP (3.4 mM) was used to generate a 'resting' background production of ADP. This resting metabolic rate was 200% higher than the quiescent rate while [NADH] and deltapsi were reduced. Subsequent ATPase additions (1.3IU.ml(-)) were made with varying amounts of Ca(2+)(0 to 535 nM) to simulate step increases in cardiac work. Ca(2+) additions increased mVo2 and depolarized deltapsi, and were consistent with an activation of Fo/F1)ATPase. In contrast, Ca(2+) reduced the [NADH] response to the ATPase addition, consistent with Ca(2+)-sensitive dehydrogenase activity (CaDH). The calculated free ADP response to ATPase decreased \2-fold in the presence of Ca(2+). The addition of 172nM free Ca(2+)] ATPase increased mVo2 by 300% (P<0.05, n=8) while deltapsi decreased by 14.9+/-0.1 mV without changes in [NADH] (P > or =0.05, n=8), consistent with working heart preparations. The addition of Ca(2+) and ATPase combined increased the mitochondrial ATP production rate with changes in deltapsi, NADH and [ADP], consistent with an activation of CaDH and F o /F(1)ATPase activity. These balancing effects of ATPase activity and [Ca(2+)] may explain several aspects of metabolic regulation in the heart during work transitions in vivo.     

Calcium ion-dependent adenosine triphosphatase activity and plasma-membrane phosphorylation in the human neutrophil.

A plasma-membrane fraction was isolated from a post-nuclear extract of human neutrophils by centrifugation through a linear sucrose density gradient. This fraction exhibited a Ca2+-dependent adenosine triphosphatase (ATPase) activity that could be differentiated from mitochondrial or myosin ATPase and from plasma-membrane Mg2+-dependent ATPase. When assayed in the presence of [gamma-32P]ATP, the Ca2+-dependent ATPase reaction resulted in the formation of an acid-resistant hydroxylamine-sensitive bond between the gamma-[32P] phosphate group and a membrane protein subunit with an apparent mol.wt. of 135000. Half-maximal activating effect of Ca2+ was found at 82nM and 0.18 microM for the ATPase and the formation of the 32P-membrane complex respectively. Generation of the phosphorylated product attained the steady state at 0 degrees C by about 30s, and was rapidly reversed by ADP. These results suggest that the Ca2+-activated ATPase reaction occurs through the formation of a phosphoprotein intermediate, similar to that described for some Ca2+-dependent ATPase enzymes associated with Ca2+ transport. The possibility thus exists that the neutrophil Ca2+-dependent ATPase catalyses a process of Ca2+ extrusion from the cell, thereby participating in the regulation of several Ca2+-dependent neutrophil functions.     

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.     

A Plasma Membrane-Type Ca2+-ATPase of 120 Kilodaltons on the Endoplasmic Reticulum from Carrot (Daucus carota) Cells (Properties of the Phosphorylated Intermediate)

Cytosolic Ca2+ levels are regulated in part by Ca2+-pumping ATPases that export Ca2+ from the cytoplasm; however, the types and properties of Ca2+ pumps in plants are not well understood. We have characterized the kinetic properties of a 120-kD phosphoenzyme (PE) intermediate formed during the reaction cycle of a Ca2+-ATPase from suspension-cultured carrot (Daucus carota) cells. Only one Ca2+-dependent phosphoprotein was formed when carrot membrane vesicles were incubated with [[gamma]-32P]ATP (W.L. Hsieh, W.S. Pierce, and H. Sze [1991] Plant Physiol 97: 1535-1544). Formation of this 120-kD phosphoprotein was inhibited by vanadate, enhanced by La3+, and decreased by hydroxylamine, confirming its identification as an intermediate of a phosphorylated-type Ca2+-translocating ATPase. The 120-kD Ca2+-ATPase was most abundant in endoplasmic reticulum-enriched fractions, in which the Ca2+-ATPase was estimated to be 0.1% of membrane protein. Direct quantitation of Ca2+-dependent phosphoprotein was used to examine the kinetics of PE formation. PE formation exhibited a Km for Ca2+ of 1 to 2 [mu]M and a Km for ATP of 67 nM. Relative affinities of substrates, determined by competition experiments, were 0.075 [mu]M for ATP, 1 [mu]M for ADP, 100 [mu]M for ITP, and 250 [mu]M for GTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 [mu]M. Calmodulin (1 [mu]M) stimulated PE formation by 25%. The results indicate that the carrot 120-kD Ca2+-ATPase is similar but not identical to animal plasma membrane-type Ca2+- ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Ca2+ pump may reside on the cortical endoplasmic reticulum, which is thought to play a major role in anchoring the cytoskeleton and in facilitating secretion.     

ADP-ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis

ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin increased the rate of exchange of actin-bound [gamma-32P]ATP by unlabelled ATP about twofold. Increased exchange rates were observed with ATP and ATP[gamma S], much less with ADP but not with AMP or NAD. ADP-ribosylation of skeletal muscle actin reduced "basal" and Mg2+ (1 mM)-induced ATP hydrolysis by about 80%. Similar inhibition of ATP hydrolysis was observed with liver actin ADP-ribosylated by Clostridium botulinum C2 toxin. The data indicate that ADP-ribosylation of actin at Arg-177 largely affects the ATP-binding and ATPase activity.     

The role of tightly bound ADP on chloroplast ATPase

Isolated chloroplast coupling factor 1 ATPase is known to retain about 1 mol of tightly bound ADP/mol of enzyme. Some experimental results have given evidence that the bound ADP is at catalytic sites, but this view has not been supported by observations of a slow replacement of the bound ADP when CaATP or MgATP is added. The experiments reported in this paper show why a slow replacement of ADP bound at a catalytic site can occur. When coupling factor 1, labeled with tightly bound [3H]ADP, is exposed to Mg2+ or Ca2+ prior to the addition of MgATP or CaATP, a pronounced lag in the onset of ATP hydrolysis is observed, and only slow replacement of the [3H]ADP occurs. Mg2+ or Ca2+ can induce inhibition very rapidly, as if an inhibited form of the enzyme results whenever the enzyme with tightly bound ADP encounters Mg2+ or Ca2+ prior to ATP. The inhibited form can be slowly reactivated by incubation with EDTA, although some irreversible loss in activity is encountered. In contrast, when MgATP or CaATP is added to enzyme depleted of Mg2+ and Ca2+ by incubation with EDTA, a rapid onset of ATP hydrolysis occurs and most of the tightly bound [3H]ADP is released within a few seconds, as expected for binding at a catalytic site. The Mg2+-induced inhibition of both the ATPase activity and the lack of replacement of tightly bound [3H] ADP can be largely prevented by incubation with Pi under conditions favoring Pi addition to the site containing the tightly bound ADP. Our and other results can be explained if enzyme catalysis is greatly hindered when MgADP or CaADP without accompanying Pi is tightly bound at one of the three catalytic sites on the enzyme in a high affinity conformation.     

Affinity labeling of the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by adenosine triphosphopyridoxal: identification of the reactive lysyl residue

Adenosine triphosphopyridoxal (AP3PL) was used as an affinity label directed toward the ATP binding site of the Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum (SR). The reagent inhibited the ATPase activity competitively with ATP, Ki = 20 microM. Incubation of SR membranes with 100 microM AP3PL followed by treatment with NaBH4 resulted in 90% inactivation of the E-P forming activity as well as of the Ca2+-transporting activity. Adenosine di- and tetraphosphopyridoxals had similar but less pronounced effects on the Ca2+-transport system. AP3PL was bound to ATPase in a one-to-one stoichiometry in parallel with the loss of the enzymatic activities. ATP and ADP prevented the binding of AP3PL and thereby protected the enzyme from inactivation. The SR membranes were labeled with [3H]AP3PL and then digested with thermolysin in order to identify the attachment site of the affinity label. A 3H-labeled peptide (Val-Glu-Pro-Ser-His-Lys* 684-Ser-Lys) was purified to homogeneity by Sephadex LH-20 chromatography and C18-reversed phase HPLC (Lys* denotes the binding site of [3H]AP3PL). These results indicate that the SR-ATPase peptide is folded in such a manner that Lys684 and Asp351, the phosphorylation site, are located very close to each other, since the distance between the 4-formyl group reacting with Lys684 and the gamma-phosphoryl group of the ATP moiety of AP3PL is rather small.     

The stereochemical course of phosphoric residue transfer catalyzed by sarcoplasmic reticulum ATPase

The stereochemical course of the phosphoric residue transfer from ADP to water catalyzed by the (Mg2+ + Ca2+)-dependent ATPase of sarcoplasmic reticulum has been determined. For this determination, the preparation is described of ATP gamma S, stereospecifically labeled in the gamma-position with both 17O and 18O. After hydrolysis of this nucleotide, the analysis of the product inorganic [16O,17O,18O]thiophosphate showed that the reaction proceeded with retention of configuration at the gamma-phosphorus atom. This result is expected since a phosphoenzyme is well characterized for this ATPase and provides support for the hypothesis that each phosphate transfer step occurs with inversion. In this case, the formation and breakdown of the phosphoenzyme occur each with inversion leading to the retention observed for the whole reaction.     

A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.     

Characterization of the phosphorylated intermediate of the K+-translocating Kdp-ATPase from Escherichia coli

During ATP hydrolysis the K+-translocating Kdp-ATPase from Escherichia coli forms a phosphorylated intermediate as part of the catalytic cycle. The influence of effectors (K+, Na+, Mg2+, ATP, ADP) and inhibitors (vanadate, N-ethylmaleimide, bafilomycin A1) on the phosphointermediate level and on the ATPase activity was analyzed in purified wild-type enzyme (apparent Km = 10 microM) and a KdpA mutant ATPase exhibiting a lower affinity for K+ (Km = 6 mM). Based on these data we propose a minimum reaction scheme consisting of (i) a Mg2+-dependent protein kinase, (ii) a Mg2+-dependent and K+-stimulated phosphoprotein phosphatase, and (iii) a K+-independent basal phosphoprotein phosphatase. The findings of a K+-uncoupled basal activity, inhibition by high K+ concentrations, lower ATP saturation values for the phosphorylation than for the overall ATPase reaction, and presumed reversibility of the phosphoprotein formation by excess ADP indicated similarities in fundamental principles of the reaction cycle between the Kdp-ATPase and eukaryotic E1E2-ATPases. The phosphoprotein was tentatively characterized as an acylphosphate on the basis of its alkali-lability and its sensitivity to hydroxylamine. The KdpB polypeptide was identified as the phosphorylated subunit after electrophoretic separation at pH 2.4, 4 degrees C of cytoplasmic membranes or of purified ATPase labeled with [gamma-32P]ATP.     

Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na+ + K+)-activated ATPase

The Mg2+-induced low-affinity nucleotide binding by (Na+ + K+)-ATPase has been further investigated. Both heat treatment (50-65 degrees C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the Kd value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na+ + K+)-ATPase activity. Thermodynamic parameters (delta H degree and delta S degree) for the apparent affinity in the ATPase reaction (Km ATP) and for the true affinity in the binding of AdoPP[NH]P (Kd and Ki) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the Km value, which thus is smaller than the Kd value. Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the Kd value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and AMP least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the Kd value. The nucleotide specificity of the low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P greater than ATP greater than ADP greater than AMP. The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the Kd value is not increased more than by ouabain alone. It is inferred that the low-affinity site is located on the enzyme, more specifically its alpha-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.     

Guanosine triphosphate utilization by canine cardiac muscle sarcoplasmic reticulum

We investigated the reaction mechanism for GTP-dependent Ca2+ uptake by canine cardiac microsomes enriched in fragmented sarcoplasmic reticulum (SR), because previous studies reported that GTP utilization in cardiac SR occurs via a pathway very different from that for ATP utilization (for a review, see "Entman, M.L., Bick, R., Chu, A., Van Winkle, W.B., & Tate, C.A. (1986) J. Mol. Cell. Cardiol. 18, 781-792"). In cardiac microsomes, we detected slow but distinct oxalate-dependent Ca2+ accumulation, which reached 550 nmol/mg protein in 10 min, and similarly slow Ca2+-dependent GTP hydrolysis. In 50 microM [gamma-32P]-GTP at 0 degrees C, we detected Ca2+-dependent formation of phosphoprotein whose level in the steady state was about a half of the maximum obtained with [gamma-32P]ATP. Kinetic properties of the phosphoprotein, its molecular weight and its chemical stability after the acid treatment are consistent with the conclusion that the phosphoprotein is an acylphosphate intermediate for Ca2+-dependent GTP hydrolysis catalyzed by the Ca2+-pump ATPase. Analysis of the kinetics of the turnover of phosphoprotein revealed that slow GTP hydrolysis is due to slow phosphoprotein formation; at 25 degrees C, the latter arises mainly from slow binding of Ca2+ to the dephosphorylated enzyme. These results indicate that, contrary to the previous data, the reaction pathway for GTP-dependent Ca2+ transport in cardiac SR is basically the same as that for ATP-dependent transport.     

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.     

Reaction mechanism of calcium-ATPase of sarcoplasmic reticulum. Substrates for phosphorylation reaction and back reaction, and further resolution of phosphorylated intermediates

Reaction of the purified Ca2+-ATPase of sarcoplasmic reticulum at 0 degrees C at low [gamma-32P]ATP (0.1 to 0.67 microM) and enzyme (0.025 to 0.24 microM) concentration in the presence of 0.11 to 30 mM Ca2+ without added Mg2+ has resulted in the formation of phosphorylated intermediate (EP:maximal level of EP = 0.45 mol/mol of enzyme) at a very slow rate. Under these conditions, the reaction steps in which EP decomposition takes place are completely prevented. This has permitted us to study the EP formation reaction and its reversal specifically, with a considerably improved time resolution. An apparent rate constant of EP formation (Vf) increases in parallel with the concentration of Ca . ATP, but not with those of Mg . ATP, or of protonated or fully ionized free ATP. This suggests that Ca . ATP is the substrate under these conditions. If Co2+ or Mn2+ are in excess over the other ions during the reaction, Vf varies in parallel with [Co . ATP] or [Mn . ATP]. Thus, it appears that either Ca2+, Co2+, or Mn2+ can be complexed with ATP to form the effective substrate. An apparent rate constant of the back reaction of EP initiated by addition of ADP to EP (Vr) increases in proportion to [ADP] or [H . ADP], but is inhibited by increasing concentrations of the ADP complex with Ca2+ or Mg2+, indicating that free ADP or protonated ADP, or both, are actual substrates for the back reaction of EP. These results suggest a new type of site to which the metal moiety of metal . ATP complex remains bound after the release of ADP from the enzyme. An acid-stable phosphorylated intermediate (EP) produced in the presence of high Ca2+ concentrations (e.g. 0.11 mM) without added Mg2+ does not decompose spontaneously, and the major portion (approximately 90%) of this EP (EPD+) reacts with ADP to form ATP (ADP-sensitive). Upon chelating Ca2+ with ethylene glycol bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), EPD+ is converted to another form of EP (EPD-), which is unreactive with ADP (or ADP-insensitive). Addition of Mg2+, after initiation of the reaction leading to EPD- by EGTA, results in rapid production of Pi from a portion of EPD- with KMg approximately equal to 3.3 x 10(3) M-1. The fraction of EPD- that is Mg2+-sensitive (EPD-,M+) increases with reaction time at a much slower rate than the Mg2+-insensitive portion of EPD- (EPD-,M-). These results suggest that the enzyme reaction involves the sequential formation of at least three forms of acid-stable EP, viz. in the order of formation, EPD+, EPD-,M-, and EPD-,M+. The equilibrium between EPD+ and EPD-,M- is shifted by higher [K+] and [Ca2+] towards EPD+.