Effects of lipids and long-chain alkyl derivatives on the activity of (Ca2+-Mg2+)-ATPase

The ATPase activity of the (Ca2+-Mg2+)-ATPase reconstituted into bilayers of phosphatidylcholines depends on the fatty acyl chain length of the phospholipids. It is shown that the fluorescence response to Ca2+ of the ATPase modified with fluorescein isothiocyanate is also dependent on phospholipid structure and is interpreted in terms of a change in the equilibrium between two forms of the ATPase, E1 and E2. A kinetic scheme for the ATPase is presented in which ATPase activity is markedly dependent on the rate of the transition between two phosphorylated forms of the ATPase, E1'PCa2 and E2'PCa2, and it is postulated that changing the phospholipid structure changes this rate. The rate of dephosphorylation of the ATPase and the ATP dependence of the E1'PCa2-E2'PCa2 transition are also lipid dependent. Binding of oleyl alcohol causes large, lipid-dependent changes in ATPase activity, and these are interpreted in terms of changes in the rates of these same steps. Oleylamine, which has been shown to bind more strongly at annular sites than at nonannular sites, inhibits ATPase activity irrespective of lipid structure, whereas fatty acids, which bind less strongly at annular sites, only inhibit at high concentrations. Methyl oleate, which binds more strongly at nonannular sites than at annular sites, causes marked stimulation for the ATPase reconstituted with short-chain lipids.     

The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca2+,Mg(2+)-ATPase.

2,5-Di(tert-butyl)-1,4-benzohydroquinone has been shown to inhibit the Ca2+,M(2+)-ATPase of sarcoplasmic reticulum with an affinity of 0.4 microM. It has been shown to shift the E2-E1 equilibrium for the ATPase towards E2, as shown previously for the inhibitor thapsigargin. The shift towards E2 results in a decrease in affinity for Ca2+, as also observed for thapsigargin. A marked decrease in the rate of the E2-E1 transition is observed for both BHQ and thapsigargin. A decrease in the equilibrium level of phosphorylation by Pi and of the steady-state level of phosphorylation by ATP are consistent with a decrease in the equilibrium constant for phosphorylation by Pi and an increase in the rate of dephosphorylation.     

Effects of Mg2+, anions and cations on the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

In a previous paper [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227] we presented a kinetic model for the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum. Here we extend the model to account for the effects on ATPase activity of Mg2+, cations and anions. We find that Mg2+ concentrations in the millimolar range inhibit ATPase activity, which we attribute to competition between Mg2+ and MgATP for binding to the nucleotide-binding site on the E1 and E2 conformations of the ATPase and on the phosphorylated forms of the ATPase. Competition is also suggested between Mg2+ and MgADP for binding to the phosphorylated form of the ATPase. ATPase activity is increased by low concentrations of K+, Na+ and NH4+, but inhibited by higher concentrations. It is proposed that these effects follow from an increase in the rate of dephosphorylation but a decrease in the rate of the conformational transition E1'PCa2-E2'PCa2 with increasing cation concentration. Li+ and choline+ decrease ATPase activity. Anions also decrease ATPase activity, the effects of I- and SCN- being more marked than that of Cl-. These effects are attributed to binding at the nucleotide-binding site, with a decrease in binding affinity and an increase in 'off' rate constant for the nucleotide.     

The mechanism of inhibition of the Ca2+-ATPase by mastoparan. Mastoparan abolishes cooperative ca2+ binding.

The amphiphilic peptide mastoparan, isolated from wasp venom, is a potent inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. At pH 7. 2, ATPase activity is inhibited with an inhibitory constant (Ki) of 1 +/- 0.13 microM. Mastoparan shifts the E2-E1 equilibrium toward E1 and may affect the regulatory ATP binding site. The peptide also decreases the affinity of the ATPase for Ca2+ and abolishes the cooperativity of Ca2+ binding. In the presence of mastoparan, the two Ca2+ ions bind independently of one another. Our results appear to support the model that describes the relationship between the two Ca2+ binding sites as "side-by-side," because this model allows the possibility of independent Ca2+ entry to the two sites. Mastoparan shifts the steady-state equilibrium between E1'Ca2 and E1'Ca2.P toward E1'Ca2.P, by possibly affecting the conformational change that follows ATP binding. The peptide also causes a reduction in the levels of phosphoenzyme formed from [32P]Pi. Some analogues of mastoparan were also tested and were found to cause inhibition of the Ca2+-ATPase in the range of 2-4 microM. The inhibitory action of mastoparan and its analogues appears dependent on their ability to form alpha-helices in membranes.     

A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1'PCa2 and to E2 modulate the rates of the transport step E1'PCa-E2'PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.     

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of activation of the plasma membrane Ca2+-ATPase by ATP and acidic phospholipids

The activation of purified and phospholipid-depleted plasma membrane Ca2+-ATPase by phospholipids and ATP was studied. Enzyme activity increased with [ATP] along biphasic curves representing the sum of two Michaelis-Menten equations. Acidic phospholipids (phosphatidylinositol (PI) and phosphatidylserine (PS)) increased Vmax without affecting apparent affinities of the ATP sites. In the presence of 20 microm ATP, phosphorylation of the enzyme preincubated with Ca2+ (CaE1) was very fast (kapp congruent with 400 s-1). vo of phosphorylation of CaE1 increased with [ATP] along a Michaelis-Menten curve (Km of 15 microm) and was phospholipid-independent. Without Ca2+ preincubation (E1 + E2), vo of phosphorylation was also phospholipid-independent, but was slower and increased with [ATP] along biphasic curves. The high affinity component reflected rapid phosphorylation of CaE1, the low affinity component the E2 --> E1 shift, which accelerated to a rate higher than that of the ATPase activity when ATP was bound to the regulatory site. Dephosphorylation of EP did not occur without ATP. Dephosphorylation increased along a biphasic curve with increasing [ATP], showing that ATP accelerated dephosphorylation independently of phospholipid. PI, but not phosphatidylethanolamine (PE), accelerated dephosphorylation even in the absence of ATP. kapp for dephosphorylation was 57 s-1 at 0 microM ATP; that rate was further increased by ATP. Steady-state [EP] x kapp for dephosphorylation varied with [ATP], and matched the Ca2+-ATPase activity measured under the same conditions. Apparently, the catalytic cycle is rate-limited by dephosphorylation. Acidic phospholipids stimulate Ca2+-ATPase activity by accelerating dephosphorylation, while ATP accelerates both dephosphorylation and the conformational change from E2 to E1, further stimulating the ATPase activity.     

Dependence on Ca2+ and tropomyosin of the actin-activated ATPase activity of phosphorylated gizzard myosin in the presence of low concentrations of Mg2+

Ca2+ and tropomyosin are required for activation of ATPase activity of phosphorylated gizzard myosin by gizzard actin at less than 1 mM Mg2+, relatively low Ca2+ concentrations (1 microM), producing half-maximal activation. At higher concentrations, Mg2+ will replace Ca2+, 4 mM Mg2+ increasing activity to the same extent as does Ca2+ and abolishing the Ca2+ dependence. Above about 1 mM Mg2+, tropomyosin is no longer required for activation by actin, activity being dependent on Ca2+ between 1 and 4 mM Mg2+, but independent of [Ca2+] above 4 mM Mg2+. Phosphorylation of the 20,000-Da light chain of gizzard myosin is required for activation of ATPase activity by actin from chicken gizzard or rabbit skeletal muscle at all concentrations of Mg2+ employed. The effect of adding or removing Ca2+ is fully reversible and cannot be attributed either to irreversible inactivation of actin or myosin or to dephosphorylation. After preincubating in the absence of Ca2+, activity is restored either by adding micromolar concentrations of this cation or by raising the concentration of Mg2+ to 8 mM. Similarly, the inhibition found in the absence of tropomyosin is fully reversed by subsequent addition of this protein. Replacing gizzard actin with skeletal actin alters the pattern of activation by Ca2+ at concentrations of Mg2+ less than 1 mM. Full activation is obtained with or without Ca2+ in the presence of tropomyosin, while in its absence Ca2+ is required but produces only partial activation. Without tropomyosin, the range of Mg2+ concentrations over which activity is Ca2+-dependent is restricted to lower values with skeletal than with gizzard actin. The activity of skeletal muscle myosin is activated by the gizzard actin-tropomyosin complex without Ca2+, although Ca2+ slightly increases activity. The Ca2+ sensitivity of reconstituted gizzard actomyosin is partially retained by hybrid actomyosin containing gizzard myosin and skeletal actin, but less Ca2+ dependence is retained in the hybrid containing skeletal myosin and gizzard actin.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 3 isoforms by steady-state and transient kinetic analyses

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca(2+)-transport cycle mediated by the human sarco(endo)plasmic reticulum Ca(2+)-ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca(2+) in activation of the overall reaction due to a decreased E(2) to E(1)Ca(2) transition rate and an increased rate of Ca(2+) dissociation from E(1)Ca(2). At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca(2+) level (either an enhanced efflux of lumenal Ca(2+) through the pump in E(2) form or insensitivity to inhibition by lumenal Ca(2+)). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca(2+) and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E(2)P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.     

GM1对肌质网Ca~(2+)-ATPase活性及膜流动性的影响

外源性GM1对肌质网Ca2+-ATPase的水解及转运活性都有明显的抑制作用.在GM1浓度为0～8nmol/mg蛋白质范围内抑制作用具有浓度依赖性.当GM1浓度达到8nmol/mg蛋白质时,酶活性受到最大抑制,此时水解活性降低51%,转运活性降低49%.荧光偏振测定结果表明:GM1参入后,肌质网膜流动性降低.     

The effect of high pressure on the conformation, interactions and activity of the Ca(2+)-ATPase of sarcoplasmic reticulum.

High pressure (100-150 MPa) increases the intensity and polarization of fluorescence of FITC-labeled Ca(2+)-ATPase in a medium containing 0.1 mM Ca2+, suggesting a reversible pressure-induced transition from the E1 into an E2-like state with dissociation of ATPase oligomers. Under similar conditions but using unlabeled sarcoplasmic reticulum vesicles, high pressure caused the reversible release of Ca2+ from the high-affinity Ca2+ sites of Ca(2+)-ATPase, as indicated by changes in the fluorescence of the Ca2+ indicator, Fluo-3; this was accompanied by reversible inhibition of the Ca(2+)-stimulated ATPase activity measured in a coupled enzyme system of pyruvate kinase and lactate dehydrogenase, and by redistribution of Prodan in the lipid phase of the membrane, as shown by marked changes in its fluorescence emission characteristics. In a Ca(2+)-free medium where the equilibrium favors the E2 conformation of Ca(2+)-ATPase the fluorescence intensity of FITC-ATPase was not affected or only slightly reduced by high pressure. The enhancement of TNP-AMP fluorescence by 100 mM inorganic phosphate in the presence of EGTA and 20% dimethylsulfoxide was essentially unaffected by 150 MPa pressure at pH 6.0 and was only slightly reduced at pH 8.0. As the enhancement of TNP-AMP fluorescence by Pi is associated with the Mg(2+)-dependent phosphorylation of the enzyme and the formation of Mg.E2-P intermediate, it appears that the reactions of Ca(2+)-ATPase associated with the E2 state are relatively insensitive to high pressure. These observations suggest that high pressure stabilizes the enzyme in an E2-like state characterized by low reactivity with ATP and Ca2+ and high reactivity with Pi. The transition from the E1 to the E2-like state involves a decrease in the effective volume of Ca(2+)-ATPase.     

Biphasic kinetics of sarcoplasmic reticulum Ca2+-ATPase and the detergent-solubilized monomer

The mechanism of ATP hydrolysis was studied at 0 degrees C and pH 7.5 using purified leaky vesicles of sarcoplasmic reticulum Ca2+-ATPase and enzyme solubilized in monomeric form with high concentrations of octaethylene glycol monododecyl ether (C12E8). The enzyme reaction of membranous Ca2+-ATPase was characterized by an initial burst in the hydrolysis of ATP and modulated by millimolar concentrations of ATP. For detergent-solubilized Ca2+-ATPase no burst and moderate low affinity modulation was observed, but the reaction was activated both at low (phosphorylating) and intermediate (K0.5 = 0.06 mM) ATP concentrations. A study of the partial reactions indicated that the effects of detergent and ATP were attributable to activation of the E1P----E2P transition which was rate-limiting. E32P dephosphorylation of membranous Ca2+-ATPase and the detergent-solubilized monomer comprised both a slow and a rapid component. The inhibitory effect of high Ca2+ was correlated with the development of a dominant contribution of slow phase dephosphorylation and with ATP-induced extra binding of Ca2+ binding which presumably takes place at the phosphorylation site (ECaP). Ca2+ was bound with lesser affinity to detergent-solubilized Ca2+-ATPase but with qualitatively the same characteristics as to membranous ECaP. Either Ca2+ or Mg2+ was required for dephosphorylation, also after detergent solubilization. It is concluded that ATP hydrolysis occurs by the same steps for membranous and monomeric Ca2+-ATPase and involves formation of either EMgP or ECaP as reaction intermediates, leading to biphasic kinetics, which, therefore, cannot be taken as evidence of an oligomeric function of the enzyme.     

Effects of Ca2+, Mg2+ and calmodulin on the formation and decomposition of the phosphorylated intermediate of the erythrocyte Ca2+-stimulated ATPase.

Formation of the phosphorylated intermediate (ECaP) of the human erythrocyte Ca2+-stimulated ATPase (Ca2+-ATPase) was more rapid and reached steady state sooner at 400 microM-Ca2+ than at 1 microM-Ca2+. Calmodulin increased the apparent rate of ECaP formation at 1 microM-Ca2+, whereas at 400 microM-Ca2+, calmodulin decreased the steady-state level of the ECaP without affecting its apparent rate of formation. Removal of endogenous Mg2+ with trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid, which decreased both the velocity and Ca2+-sensitivity of the Ca2+-ATPase, did not alter the Ca2+-sensitivity or the apparent rate of formation of ECaP. ECaP formation at high Ca2+ concentrations was not affected by Mg2+ concentrations as high as 1 mM, and the ECaP could be dephosphorylated by ADP and ATP along either the forward or reverse pathways. The results suggest that high Ca2+ concentrations inhibit Ca2+-ATPase activity by preventing dephosphorylation of the E2P complex, rather than by inhibition of the transformation from E1CaP ('high-Ca2+-affinity' ECaP) to E2CaP ('lower-energy' ECaP).     

Kinetic characterization of the normal and detergent-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump. Rate-limiting step(s) under different conditions

We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and M?ller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.     

Modulatory ATP binding affinity in intermediate states of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase

The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca(2+)-ATPase displays an ATP affinity of 4 μM for the E2P ground state analog, 1 μM for the E2P transition state and product state analogs, and 11 μM for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe(487) (N-domain) with serine, Arg(560) (N-domain) with leucine, or Arg(174) (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile(188) (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile(188) in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys(205) (A-domain) or Glu(439) (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca(2)E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca(2+)-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.     

Characterization of phenylmaleimide inhibition of the Ca(2+)-ATPase from skeletal-muscle sarcoplasmic reticulum

The Ca(2+)-ATPase from sarcoplasmic reticulum reacts with phenylmaleimide, producing the inhibition of the ATPase activity following a pseudo-first-order kinetic with a rate constant of 19 M(-1) s(-1). Calcium and ATP binding are not altered upon phenylmaleimide inhibition. However, the presence of millimolar calcium, and to a lesser extent magnesium, in the inhibition medium enhances the effect of phenylmaleimide, causing a higher degree of inhibition. Solubilization with C(12)E(8) does not affect the ATPase inhibition, excluding any kind of participation of the lipid bilayer. Phosphorylation with ATP in steady-state conditions as well as phosphorylation with inorganic phosphate in equilibrium conditions were strongly inhibited. Conversely, we have found that the occupancy of the phosphorylation site by ortovanadate fully protects against the inhibitory effect of phenylmaleimide, indicating a conformational transition associated with the phosphorylation reaction.     

Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca2+-ATPase at low water content: water dependence of the E2-->E1 conversion.

Enzymes entrapped in reverse micelles can be studied in low-water environments that have the potential of restricting conformational mobility in specific steps of the reaction cycle. Sarcoplasmic reticulum Ca2+-ATPase was incorporated into a reverse-micelle system (TPT) composed of toluene, phospholipids, Triton X-100 and varying amounts of water (0.5-7%, v/v). Phosphorylation of the Ca2+-ATPase by ATP required the presence of both water and Ca2+ in the micelles. No phosphoenzyme (EP) was detected in the presence of EGTA. Phosphorylation by Pi (inorganic phosphate) in the absence of Ca2+ was observed at water content below that necessary for phosphorylation by ATP. In contrast to what is observed in a totally aqueous medium, EP formed by Pi was partially resistant to dephosphorylation by Ca2+. However, the addition of non-radioactive Pi to the EP already formed caused a rapid decrease in radiolabelled enzymes, as expected for the isotopic dilution, indicating the existence of an equilibrium (E+Pi<-->EP). Phosphorylation by Pi also occurred in TPT containing millimolar Ca2+ concentrations in a range of water concentrations (2-5% v/v). The substrates p-nitrophenyl phosphate, acetyl phosphate, ATP and GTP increased the EP level under these conditions. These results suggest that: (1) the rate of conversion of the ATPase conformer E2 into E1 is greatly reduced at low water content, so that E2-->E1 becomes the rate-limiting step of the catalytic cycle; and (2) in media of low water content, Pi can phosphorylate both E1Ca and E2. Thus, the effect of enzyme hydration is complex and involves changes in the phosphorylation reaction at the catalytic site, in the equilibrium between E2 and E1 conformers, and in their specificity for substrates.     

The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase

We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a clear preference for NaKbeta1 over NaKbeta3 and gastric HKbeta. NH4(+), K+, and Na+ maximally increased the activity of HKalpha2-NaKbeta1 to 24.0, 14.2, and 5.0 micromol P(i) x mg(-1) protein x h(-1), respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH4(+)-stimulated ATPase activity, a turnover number of 20,000 min(-1) was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4(+) > Na+, indicating that K+ is a stronger E2 promoter than NH4(+), whereas in the presence of Na+ the enzyme is in the E1 form. For K+ and NH4(+), the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4(+) can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat nongastric H,K-ATPase.     

Kinetic characterization of the perturbation by dodecylmaltoside of sarcoplasmic reticulum Ca(2+)-ATPase.

We investigated the functional aspects of the interaction between the sarcoplasmic reticulum (SR) membranous Ca(2+)-ATPase and the non-ionic detergent dodecylmaltoside, using detergent concentrations allowing perturbation of the membrane but not its solubilization. At pH 7.5, the effects of dodecylmaltoside on ATPase activity and delipidation had previously been shown to resemble, in some respects, those of octa(ethylene glycol) monododecylether (C12E8), an appropriate detergent for ATPase studies. Our aim here was to explore the specific effects of dodecylmaltoside on the different steps in the ATPase catalytic cycle, which may owe their specificity to the difference between the polar head groups of dodecylmaltoside and C12E8. This was done at 20 degrees C, both at pH 6 in the absence of KCl and at pH 7.5 in the presence of 100 mM KCl, two conditions under which the characteristics of unperturbed ATPase have already been well defined. Preliminary estimation of dodecylmaltoside partition between water and SR membranes at pH 6 yielded a partition coefficient K close to 4 x 10(5) (ratio of the molar fraction of dodecylmaltoside in the lipid to that in the aqueous phase at a low detergent concentration, assuming that most of this detergent was present in the lipid phase). At near saturation of SR membranes, bound dodecylmaltoside was roughly equimolar with the constituent phospholipids. Non-solubilizing concentrations of dodecylmaltoside inhibited SR ATPase activity by up to 65-70% at pH 7.5, but not at pH 6, unlike the results of similar experiments with C12E8. The rates of the four main steps in the ATPase catalytic cycle were measured by fast kinetic techniques; they were similarly modified at both pH. Dodecylmaltoside slowed down both the rate of calcium-saturated ATPase phosphorylation and the rate of ATPase isomerization after phosphorylation, two steps which were not targets of perturbation by C12E8. The slowing down of the isomerization step by dodecylmaltoside might well explain why it inhibited overall ATPase activity at pH 7.5. In contrast to C12E8, dodecylmaltoside did not affect the dephosphorylation step, which was the main target of inhibition by C12E8 and the main rate-limiting step at pH 6. However, like C12E8, dodecylmaltoside accelerated the calcium binding-induced transition of nonphosphorylated ATPase. Another striking feature of the perturbation induced by dodecylmaltoside was that it significantly altered the binding of 45Ca2+ to the ATPase and the corresponding conformational changes. At pCa 5-5.5, it almost halved calcium binding to the ATPase but ATPase phosphorylation was unimpaired.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a global inhibitor-induced conformation change on the Ca(2+)-ATPase of sarcoplasmic reticulum from paired inhibitor studies

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by a variety of hydrophobic, hydroxy-containing molecules. A kinetic method has been used to study competition between binding of pairs of inhibitors to the ATPase. The presence of 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ) decreases the affinity of the ATPase for 2,5-dipropyl-1,4-dihydroxybenzene (PHQ), suggesting that PHQ and BHQ bind to the same site on the ATPase. In contrast, the presence of BHQ increases the affinity of the ATPase for curcumin and vice versa. This suggests that BHQ and curcumin bind to separate sites on the ATPase and that binding of the first inhibitor to the ATPase results in a change to a conformation with higher affinity for the second inhibitor. This is consistent with previous experiments with BHQ and thapsigargin suggesting a conformation change on inhibitor binding, E2 + I <--> 2; E2I <--> 2; E2(A)I, with E2(A)I having a higher affinity for the second inhibitor than E2. The affinity for BHQ is also increased by binding of diethylstilbesterol, ellagic acid, or nonylphenol, and the affinity for curcumin is also increased by ellagic acid. These results showing that binding of a variety of inhibitors of very different structures all result in a general increase in inhibitor affinity point to a global conformational change on the Ca(2+)-ATPase caused by inhibitor binding, as well as any local, inhibitor-specific changes in conformation.