How do MgATP analogues differentially modify high-affinity and low-affinity ATP binding sites of Na+/K(+)-ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.     

Separation of active and inactive (nonphosphorylating) Ca(2+)-ATPase in sarcoplasmic reticulum subfractions from low-frequency-stimulated rabbit muscle.

Chronic low-frequency stimulation elicits in rabbit fast-twitch muscle a partial inactivation of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Ca(2+)-uptake activities. Inactive Ca(2+)-ATPase was enriched in a light microsomal fraction by sucrose density gradient centrifugation after calcium oxalate loading in the presence of ATP. This fraction showed a reduced specific activity and phosphoprotein formation of the Ca(2+)-transport ATPase. These results suggest that the inactivation of the Ca(2+)-ATPase as induced by increased contractile activity, is confined to a specific SR vesicle population.     

Fluoroaluminate complexes are bifunctional analogues of phosphate in sarcoplasmic reticulum Ca(2+)-ATPase.

The mechanism of inhibition of the sarcoplamc reticulum (SR) Ca(2+)-ATPase by the fluoroaluminate complexes was investigated. First, AlF4- was shown to bind to the Ca(2+)-free conformation of the enzyme by a slow quasi-irreversible process. The rate constants of the reaction are k+ = 16 x 10(3) M-1 s-1 and k- < 1.5 10(-3) s-1. We directly measured a stoichiometry of about 4.8 nmol of AlF4- bound/mg of protein. Mg2+ was a necessary cofactor for the reaction with a dissociation constant of 3 mM. It was demonstrated (Dupont, Y., and Pougeois, R. (1983) FEBS Lett. 156, 93-98) that phosphorylation by P(i) induced a dehydration of the catalytic site. The same process has been shown here to occur upon AlF4- binding either by the use of Me2SO or by demonstration of an increase of bound 2',3'-O-(2,4,6-trinitrocyclohexadienyldene)adenosine triphosphate fluorescence. Phosphorylation by P(i) is inhibited by the binding of AlF4-. Second, a fluoroaluminate complex, presumably AlF4-, was also shown to bind to the Ca(2+)-bound conformation of the Ca(2+)-ATPase in the presence of ADP and stabilize a E1.Ca2.ADP.AlFx complex. The dissociation constant of the nucleotidic site for ADP was shifted to the micromolar range. The Ca2+ ions bound on the external high affinity sites became occluded upon binding of (ADP + AlFx). We propose that AlF4- mimics P(i) binding to the Ca(2+)-free conformation of the ATPase and stabilizes an intermediate similar to the acyl-phosphate derivative; it also acts as an analogue of the gamma-phosphate of ATP and stabilizes an E1.[Ca2].ADP.AlF4 complex where the Ca2+ ions are occluded.     

Interaction of adenosine-5'-O-(3-thiotriphosphate) with Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum

Interaction of adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) with Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. The nucleotide was slowly hydrolyzed by the ATPase at 30 degrees C at a rate of about 0.5% that of ATP hydrolysis. Whereas at 0 degrees C, ATP gamma S showed only a limited reactivity toward the ATPase in that a thiophosphorylated intermediate was formed and ADP was released, but hydrolysis of the intermediate to complete the catalytic cycle did not occur. A fairly stable analog of the E-P intermediate could thus be obtained. Presence of the thiophosphorylated intermediate was indicated by the [3H]ADP in equilibrium ATP gamma S exchange reaction and also by using [35S]ATP gamma S. When the ATPase was reacted with ATP gamma S at 0 degrees C in the presence of ferricyanide, EP-forming activity was rapidly lost. Free Ca2+ ions were required for this inactivation. Disulfide bond formation between a cysteinyl residue located near the substrate binding site and the enzyme-bound ATP gamma S or the thiophosphorylated intermediate was suggested by the fact that 2-mercaptoethanol reversed the inactivation. The reaction may prove to be a useful tool for affinity labeling of the active site of the ATPase.     

Effect of ovarian steroids on membrane ATPase activities in microsomes (microsomal fractions) from rat myometrium. Inhibition of a component of the Mg2+-activated ATPase by Ca2+-calmodulin and by oxytocin.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.     

Fe(2+)-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg(2+) and Mg(2+)-ATP sites

Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.     

Transient kinetic analysis of turnover-dependent fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP bound to Ca2+-ATPase of sarcoplasmic reticulum

The fluorescence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is greatly enhanced during turnover induced by ATP plus Ca2+ (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). We have studied the kinetics of induction of TNP-ATP fluorescence and of its decay and have found a close correlation with levels of phosphorylated intermediate of the enzyme, E-P. Steady-state kinetic studies suggested competitive binding of ATP and TNP-ATP to the catalytic site, with Km and Ki values of 2.4 and 1.0 microM, respectively. Rate constants for fluorescence enhancement and for E-P formation in the presteady state were 1.2 s-1 or 97-130 s-1 under conditions resulting in TNP-ATP or ATP saturation respectively, of the enzyme at inception of reaction. The slow process was concluded to be the koff for dissociation of TNP-ATP from the catalytic site. Following this dissociation, a second TNP-ATP site was detected, which both formed (97-130 s-1) and decayed (0.22 s-1) synchronously with E-P. TNP-ATP binding to this noncatalytic site was rapid (5 X 10(7) M-1 s-1) and resulted in high fluorescence during steady-state turnover. Fluorescence was found to be dissociated from E-P by KCl (100 mM). KCl had little effect on E-P levels, but decreased fluorescence by 68%. These studies provide independent kinetic evidence for the existence of both catalytic and noncatalytic, or "regulatory," nucleotide-binding sites, but cannot distinguish whether the two sites exist independently or whether the catalytic site is transformed into a regulatory site on phosphorylation. The latter site, which shows relatively high selectivity for TNP-ATP over ATP, and which is simultaneously hydrophobic and freely accessible to the medium, may play a role during energy transduction. The changes occurring at this site during catalysis are conveniently monitored with TNP-ATP fluorescence.     

The presence of sarcolipin results in increased heat production by Ca(2+)-ATPase

Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane alpha-helix. When reconstituted with the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca(2+). Heats of reaction of the reconstituted Ca(2+)-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca(2+)-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN.ATPase complex of 6.9 x 10(-4) +/- 2.9 x 10(-4) in units of mol fraction per monolayer. It is suggested that the interaction between Ca(2+)-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum.     

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

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

Interaction of an aromatic dibromoisothiouronium derivative with the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum

Isothiouronium compounds [Hoving, S., Bar-Shimon, M., Tijmes, J. J., Goldshleger, R., Tal, D. M., and Karlish, S. J. (1995) J. Biol. Chem. 270, 29788-29793] act as high-affinity competitive antagonists for Na(+) and K(+) (Rb(+)) on the renal Na(+)/K(+)-ATPase where they favor the E1 conformation. We have now characterized the effects of 1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene (Br(2)-TITU) on the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum. Br(2)-TITU inhibited the Ca(2+)-ATPase, both transport and catalytic activity, with a K(0.5) of 5-15 microM. Maximum inhibition was at 10 min with t(0.5) of 3-5 min. Br(2)-TITU, 100 microM, quenched Trp autofluorescence by 80%, but the residual signal still responded to Ca(2+) binding. Maximum quenching of fluorescence was at pH 9.0. Total E-P levels, during the steady state of turnover of the Ca(2+)-ATPase, were increased from 0.5 to 5.8 nmol x mg(-1) by Br(2)-TITU at pH 6.8. Trinitrophenyl-ATP (TNP-ATP) superfluorescence, which monitors hydrophobicity of the ATP site, was increased 3-4-fold, suggesting that Br(2)-TITU favors an "E2"-like state. Fluorescence was also increased 3-5-fold when E-P was induced with P(i) plus EGTA. Br(2)-TITU increased the rate constants of induction of superfluorescence with ATP plus Ca(2+) from 0.32 to 0.69 s(-1) and with P(i) plus EGTA from 0.84 to 7.45 s(-1). Br(2)-TITU also decreased rate constants for "off" reactions from 2.9 to 0.66 s(-1) and from 10.9 to 0.73 s(-1) for the ATP and P(i) reactions, respectively. Br(2)-TITU, which competitively inhibits the Na(+)/K(+)-ATPase, has a novel effect on the Ca(2+)-ATPase. It promotes accumulation of E2-P species due to increased rate of formation and decreased rate of hydrolysis and quenches tryptophan autofluorescence. Br(2)-TITU could be a useful inhibitor to probe intermediate reactions of the Ca(2+)-ATPase that link catalysis with Ca(2+) translocation.     

Interdependence of Ca2+ occlusion sites in the unphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase complex with CrATP.

The beta, gamma-bidentate chromium(III) complex of ATP (CrATP) was used as a substrate analog to stabilize a form of the Ca(2+)-ATPase of the sarcoplasmic reticulum containing both of the bound calcium ions in an occluded state without enzyme phosphorylation. The kinetics of dissociation of Ca2+ from the occlusion sites in the CrATP-enzyme complex were consistent with the existence of two nonequivalent and interdependent Ca2+ occlusion sites, both in the membranous Ca(2+)-ATPase and in a detergent-solubilized monomeric Ca(2+)-ATPase preparation. The rate constant for release of the first calcium ion was k1 = 0.99 h-1, whereas the second calcium ion was released with a rate constant of k2 = 0.25 h-1 when the first site was empty and with a rate constant of k3 = 0.13 h-1 when the first site was occupied by Ca2+. Ca2+ binding at the first site occurred with a rate constant of k-1 = 0.96 microM-1 h-1 (apparent Kd = 1.0 microM). The Ca(2+)-occluded state was further stabilized by ADP, binding in exchange with ATP with an apparent Kd of 8.6 microM. Two kinetic classes of CrATP-binding sites were observed, each with a stoichiometry of 3-4 nmol/mg of protein; but only the fast phase of CrATP binding was associated with Ca2+ occlusion. Derivatization of the Ca(2+)-ATPase with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodimide resulted in inactivation of phosphorylation of the enzyme from MgATP, whereas the ability to occlude Ca2+ in the presence of CrATP was retained, albeit with a reduced apparent affinity for Ca2+.     

Early steps of the Mg(2+)-ATPase of relaxed myofibrils. A comparison with Ca(2+)-activated myofibrils and myosin subfragment 1.

The early steps of the Mg(2+)-ATPase activity of relaxed rabbit psoas myofibrils were studied in a buffer of near-physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase, and the cleavage and release of product steps by the Pi burst method. The data obtained were interpreted by [formula: see text] where M represents the myosin heads with or without actin interaction. This work is a continuation of our study on Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. Here the constants obtained with relaxed myofibrils were compared with those with activated myofibrils and myosin subfragment 1 (S1). We find that whereas Ca2+ increases 80X the release of products (k4), it has little effect upon the kinetics of the initial binding and cleavage steps. As with activated myofibrils and S1, the second-order binding constant for ATP (k2/K1) was about 1 microM-1 s-1 and the ATP was bound very tightly. With activated myofibrils, it was difficult to obtain an estimate for the koff for ATP(k-2) but it is much less than kcat. Here with relaxed myofibrils we estimate k-2 less than 8 x 10(-4) s-1, which is considerably smaller than kcat (0.019 s-1) and also previous estimates for this constant. The overall Kd for ATP to relaxed myofibrils is less than 8 x 10(-10) M. With S1 this Kd is about 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by 2,5-di(tert-butyl)-1,4-benzohydroquinone.

We characterized the interaction of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) with the sarcoplasmic reticulum (SR) Ca(2+)-ATPase from rabbit fast-twitch skeletal and canine cardiac muscles by examining the effect of this agent on the ATPase reaction. tBuBHQ at less than 10 microM inhibited ATP hydrolysis by both isoforms of Ca(2+)-ATPase by up to 80 and 90%, respectively. The half maximal inhibition of these enzymes was observed at about 1.5 microM tBuBHQ. Thus, this agent potently inhibits the fast-twitch skeletal and slow-twitch skeletal/cardiac isoforms of SR Ca(2+)-ATPase. tBuBHQ at 5-10 microM inhibited the rate of decomposition of the phosphoenzyme intermediate (EP), measured as a ratio between ATPase activity and the EP level in the steady state, by 35-40%. It also inhibited formation of EP by decreasing the rate of Ca2+ binding to the Ca(2+)-deficient, nonphosphorylated enzyme to about 1/8 of the control value. These results indicate that tBuBHQ has at least two sites of action in the reaction sequence for the SR Ca(2+)-ATPase.     

Measurement of sarcoplasmic reticulum Ca2+-ATPase activity and E-type Mg2+-ATPase activity in rat heart homogenates

The presence of a high and nonlinear Ca2+-independent (or basal) ATPase activity in rat heart preparations makes difficult the reliable measurement of sarcoplasmic reticulum (SR) Ca2+-ATPase activity by usual methods. A spectrophotometric assay for the accurate determination of SR Ca2+-ATPase activity in unfractionated homogenates from rat heart is described. The procedure is based on that reported by Simonides and van Hardeveld (1990, Anal. Biochem. 191, 321-331) for skeletal muscle homogenates. To avoid overestimation of the Ca2+-ATPase activity of cardiac homogenates that occurs when sequential measurements of total and basal ATPase activities are performed, two parallel and independent assays are required: one with low (micromolar) and other high (millimolar) calcium concentration. Addition of thapsigargin (0.2 microM) blocked totally the activity considered as Ca2+-ATPase activity. Using this method, the rat heart homogenate Ca2+-ATPase activity was 10.5 +/- 2.0 micromol. min-1 x g-1 tissue wet weight (n = 8). Likewise, a spectrophotometric assay for measuring E-type Mg2+-ATPase activity in cardiac total homogenates has been developed, comparing the following characteristics of the enzymatic activity in homogenate and a membrane-enriched fraction: first-order rate constant for ATP-dependent inactivation, Km for ATP, and effects of concanavalin A, Triton X-100, and specific inhibitors.     

Characterization of the membrane bound Mg2+-ATPase of rat skeletal muscle

A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg2+-ATPase (5-25 mumol Pi/mg min). The rate of ATP hydrolysis by the Mg2+-ATPase was nonlinear but decayed exponentially (first-order rate constant greater than or equal to 0.2 s-1 at 37 degrees C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AdoPP[NH]P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or AdoPP[NH]P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg2+-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg2+-ATPase to detergents. It is proposed that the regulation of the Mg2+-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on (K+ + H+)-ATPase. I. Essential arginine residue in its substrate binding center

1. A membrane vesicle fraction containing a high (K+ + H+)-ATPase activity was isolated from porcine gastric mucosa. The enzyme has a pH optimum of 7.0 and is stimulated by T1+, K+, Rb+ and NH4+ with KA values of 0.13, 2.7, 7.6 and 26 mM, respectively, at this pH. 2. Incubation of the isolated membrane fraction with butanedione leads to inactivation of the (K+ + H+)-ATPase activity. The pH-dependence of the (K+ + H+)-ATPase activity. The pH-dependence of the inactivation and the reversibility of the reaction, observed after removal of excess butanedione and borate, indicate that modification of arginine is involved. 3. The inactivation of (K+ + H+)-ATPase activity by butanedione is time-dependent and follows second-order kinetics. From the dependence of the inactivation rate on the reagent concentration it appears that a single arginine residue is involved in the inactivation of the (K+ + H+)-ATPase activity. 4. ATP, deoxy-ATP, ADP and adenylyl imidodiphosphate (AMPPNP), but not CTP, GTP and ITP which are poor substrates, protect the enzyme against butanedione inactivation, suggesting that the essential arginine residue is located in the ATP binding centre. 5. In the presence of Mg2+ the butanedione inactivation is increased, and the protection by ATP, deoxy-ATP and ADP (but not that by AMPPNP) is less pronounced. This suggests that Mg2+ induces a conformational change in the enzyme, exposing the arginine group and coinciding with phosphorylation and subsequent release of ADP from its binding site.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

AlF4- reversibly inhibits ''P''-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.     

Chemical modification of sarcoplasmic reticulum with methylbenzimidate. Stimulation of Ca2+ efflux.

Treatment of sarcoplasmic reticulum membranes with 12 mM-methylbenzimidate (MBI) for 5 min, in the presence of 5 mM-ATP at pH 8.5, resulted in a 2-3-fold stimulation of ATP hydrolysis and over 90% inhibition of Ca2+ accumulation. This phenomenon was strictly dependent upon the presence of nucleotides with the following order of effectiveness: adenosine 5'-[beta, gamma-imido]triphosphate greater than or equal to ATP greater than UTP greater than ADP greater than AMP. Divalent cations such as Ca2+, Mg2+ and Mn2+, when present during the MBI treatment, prevented both the stimulation of ATPase activity and the inhibition of Ca2+ accumulation. Modification with MBI had no effect on E-P formation from ATP, ADP-ATP exchange, Ca2+ binding or ATP-Pi exchange catalysed by the membranes. Membranes modified with MBI in the presence of ATP and then passively loaded with Ca2+ released about 80% of their Ca2+ content within 3 s. Control membranes released only 3% of their Ca2+ during the same time period. MBI modification inhibited Ca2+ accumulation by proteoliposomes reconstituted with the partially purified ATPase but not with the purified ATPase fraction. These results suggest that MBI in the presence of ATP stimulates Ca2+ release by modifying a protein factor(s) other than the (Ca2+ + Mg2+)-ATPase.