Absorbance and fluorescence properties of 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate bound to coupled and uncoupled Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Preincubation of skeletal muscle sarcoplasmic reticulum vesicles in the presence of the calcium chelator, [ethylenebis(oxyethylenenitrilo)tetraacetic acid] (EGTA), irreversibly uncouples calcium transport from ATP hydrolysis. Uncoupling cannot be explained by increased membrane permeability, but is associated with decreased capacity of the Ca2+-ATPase to bind noncatalytic, tightly bound ATP and ADP (Berman, M. C. (1982) Biochim. Biophys. Acta 694, 95-121). The effects of EGTA-induced uncoupling on absorbance and fluorescence properties of the bound ATP analog, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), have been studied under static and turnover conditions. Binding of 4.5-4.9 nmol of TNP-ATP/mg, as determined by absorbance difference titration, was relatively unaffected in the uncoupled state. TNP-ATP, bound to coupled vesicles during turnover, showed 6-8-fold enhanced fluorescence and a shift in the difference absorbance maximum from 510 to 493 nm, indicating increased hydrophobicity of the noncatalytic site. Turnover-dependent fluorescence enhancement was diminished by 60-70% in the uncoupled state, while the absorbance maximum wavelength shift was abolished. These data, correlating changes in the environment of the noncatalytic or regulatory nucleotide binding site on the Ca2+-ATPase with coupling activity, indicate that uncoupling is an intramolecular process, involving a ligand binding site on the ATPase, and that exclusion of H2O from the site occupied by noncatalytic nucleotides, during at least part of the catalytic cycle, is an event associated with energy transduction.     

Interaction of valinomycin and monovalent cations with the (Ca2+,Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum

The interactions of monovalent cations and of the K+-specific ionophore, valinomycin, with the Ca2+-ATPase of skeletal muscle of sarcoplasmic reticulum have been studied in the absence of cation gradients by their effects on enzyme turnover and on the ATP plus Ca2+-dependent enhanced fluorescence of the ATP analogue, 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine 5'-triphosphate (TNP-ATP) (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Monovalent cations decreased turnover-dependent TNP-ATP fluorescence in the series K+ greater than Rb+ approximately equal to Cs+ greater than Na+ greater than Li+ (K0.5 = 49, 73, 75, 94, and 246 mM, respectively), consistent with the known specificity of the monovalent cation binding site that stimulates turnover and E-P hydrolysis. Valinomycin (200 nmol/mg), in the absence of monovalent cations, decreased ATPase activity by 30% and abolished the stimulatory effects of 150 mM KCl or NaCl on turnover. The ionophore alone enhanced TNP-ATP fluorescence by 20% and altered the specificity and affinity of the site that inhibited TNP-ATP fluorescence to Cs+ greater than Rb+ greater than K+ approximately equal to Na+ greater than Li+ (K0.5 = 79, 111, 134, 136, and 270 mM, respectively), which follows the Hofmeister series for effectiveness of monovalent lyotropic cations. TNP-ATP binding was not affected by either monovalent cations or valinomycin. Inhibition of turnover-dependent TNP-ATP fluorescence appears to be a useful parameter for monitoring monovalent cation binding to the Ca2+-ATPase. It is concluded that the ionophore interacts directly with the Ca2+-ATPase, independent of its K+ conductance effects on the lipid bilayer, and modifies the affinity and specificity of the monovalent cation site, either by direct interaction or by the formation of a valinomycin-monovalent cation-enzyme complex.     

Energetics of the calcium-transporting ATPase

A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.     

Phosphoenzyme conformational states and nucleotide-binding site hydrophobicity following thiol modification of the Ca2+-ATPase of sarcoplasmic reticulum from skeletal muscle

Enhanced fluorescence of the ATP analogue 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine 5'-triphosphate (TNP-ATP), bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, is closely related to phosphoenzyme levels (Bishop, J. E., Johnson, J. D., and Berman, M. C. (1984) J. Biol. Chem. 259, 15163-15171) and has an emission maximum consistent with decreased polarity of the TNP-ATP-binding site. The phosphoenzyme conformation responsible for increased nucleotide-binding site hydrophobicity has been studied by redistribution of phosphoenzyme intermediates following specific thiol group modification. N-Ethylmaleimide, in the presence of 50 microM Ca2+, 1 mM adenyl-5'-yl imidodiphosphate, pH 7.0, at 25 degrees C for 30 min, selectively modified the SH group essential for phosphoenzyme decomposition, which resulted in decreased ATPase activity, Ca2+ uptake, and a decrease in ATP-induced TNP-ATP fluorescence. Phosphorylated (Ca2+, Mg2+)-ATPase levels from [gamma-32P] ATP remained relatively unaffected (3.1 nmol/mg), but the ADP-insensitive fraction decreased from 56 to 15%. Phosphoenzyme levels from 32Pi were also decreased to the same extent as turnover, with equivalent loss of Pi-induced TNP-ATP fluorescence. The E1 to E2 transition, as monitored by the change in intrinsic tryptophan fluorescence, was unaffected. Modification of thiol groups of unknown function did not modify turnover-induced TNP-ATP fluorescence. It is concluded that the ADP-insensitive phosphoenzyme, E2-P, is responsible for enhanced TNP-ATP fluorescence. This suggests that the conformational transition, 2Ca2+outE1 approximately P----2Ca2+inE2-P, is associated with altered properties of the noncatalytic, or regulatory, nucleotide-binding site.     

Kinetic mechanism of 1-N6-etheno-2-aza-ATP hydrolysis by bovine ventricular myosin subfragment 1 and actomyosin subfragment 1. The nucleotide binding steps

The large change in fluorescence emission of 1-N6-etheno-2-aza-ATP (epsilon-aza-ATP) has been used to investigate the kinetic mechanism of etheno-aza nucleotide binding to bovine cardiac myosin subfragment 1 (myosin-S1) and actomyosin subfragment 1 (actomyosin-S1). The time course of nucleotide fluorescence enhancement observed during epsilon-aza-ATP hydrolysis is qualitatively similar to the time course of tryptophan fluorescence enhancement observed during ATP hydrolysis. In single turnover experiments, the nucleotide fluorescence rapidly increases to a maximum level, then decreases with a rate constant of 0.045 s-1 to a final level, which is about 30% of the maximal enhancement; a similar fluorescence enhancement is obtained by adding epsilon-aza-ADP to cardiac myosin-S1 or actomyosin-S1 under the same conditions (100 mM KCl, 10 mM 4-morpholinepropanesulfonic acid, 5 mM MgCl2, 0.1 mM dithiothreitol, pH 7.0, 15 degrees C). The kinetic data are consistent with a mechanism in which there are two sequential (acto)myosin-S1 nucleotide complexes with enhanced nucleotide fluorescence following epsilon-aza-ATP binding. The apparent second order rate constants of epsilon-aza-ATP binding to cardiac myosin subfragment 1 and actomyosin subfragment 1 are 2-12 times slower than those for ATP. Actin increases the rate of epsilon-aza-ADP dissociation from bovine cardiac myosin-S1 from 1.9 to 110 s-1 at 15 degrees C which can be compared to 0.3 and 65 s-1 for ADP dissociation under similar conditions. Although there are quantitative differences between the rate and equilibrium constants of epsilon-aza- and adenosine nucleotides to cardiac actomyosin-S1 and myosin-S1, the basic features of the nucleotide binding steps of the mechanism are unchanged.     

Characterization of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP

The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.     

Inhibition of sodium and potassium adenosine triphosphatase by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenine nucleotides. Implications for the structure and mechanism of the Na:K pump

Trinitrophenyl derivatives of adenine nucleotides (TNP-nucleotides: 2',3'-O-2,4,6-trinitrocyclohexadienylidene complexes at neutral or basic pH) are potent inhibitors of (Na,K)-ATPase activity. The inhibitory potency of the derivatives tested followed the sequence: TNP-ADP greater than TNP-ATP greater than TNP-AMP much greater than TNP-IMP greater than TNP-adenosine. In the presence of Na+ plus K+, high and low affinity activation of ATPase activity by ATP was observed. Under these conditions, TNP-ATP inhibited (Na,K)-ATPase activity competitively with respect to ATP at the kinetically defined "low affinity ATP site." In the presence of Na+ alone, only high affinity activation by ATP was observed. Under these conditions, TNP-ATP inhibited (Na)-ATPase and enzyme phosphorylation by competing with ATP at the kinetically defined "high affinity ATP site." The Ki values for inhibition were similar to the KD values determined by direct TNP-ATP binding measurements, indicating that the same TNP-ATP site is involved in the inhibition of (Na,K)-ATPase and (Na)-ATPase activities. We conclude that high and low affinity ATP "sites" are interconvertible (i.e. they represent two forms of the same site) and do not co-exist independently. TNP-ATP also inhibited competitively the K+-stimulated p-nitrophenyl phosphatase activity and enzyme phosphorylation by Pi, suggesting that the catalytic site for these substrates is associated with the TNP-ATP site. A kinetic model for (Na,K)-ATPase turnover based on a single ATP site which changes affinity during turnover is presented. The model was analyzed by the King-Altman (1956) J. Phys. Chem. 60, 1375-1378) method to obtain the steady state equation for the rate of ATP hydrolysis as a function of ATP concentration. Computer simulations using published values of the rate constants of intermediate steps suggest that the model is adequate to describe the observed dependence of enzyme activity on ATP concentration and the inhibition by TNP-ATP. The implications of these results on the structure and mechanism of the (Na,K) pump are discussed.     

Location of lysine-beta 162 in mitochondrial F1-adenosinetriphosphatase

The quenching of the fluorescence of bovine heart F1-adenosinetriphosphatase labeled specifically at its essential Lys-beta 162 with 7-chloro-4-nitro-2,1,3-benzoxadiazole (N-NBD-F1) by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-triphosphate (TNP-ATP) has been studied. Analysis of the fluorescence data in the presence of 1 mM ATP shows that the dissociation constant of TNP-ATP from its first binding site in the covalently labeled enzyme is 250-fold lower than that of ATP, which it replaces in pH 7.0 buffer containing 25% glycerol, and that this binding causes a 54% quenching of the fluorescence of the N-NBD label due to energy transfer to the weakly fluorescent TNP-ATP molecule. Computation based on the observed quenching gives a distance of 25.6 +/- 0.4 A between the NBD label and the chromophore of the bound TNP-ATP molecule. Since the distance between the chromophore and the farthest O atom of the bound TNP-ATP is about 16 A, it seems quite likely that the epsilon-amino group of Lys-beta 162 is near the gamma-phosphate group of the TNP-ATP bound at the catalytic site. Similar measurements in the presence of 1 mM ADP show that the replacement of ADP at the catalytic site by TNP-ATP causes a 49% quenching of the fluorescence of the N-NBD label, which gives a distance of 26.5 +/- 0.4 A between the label and the chromophore of the bound TNP-ATP molecule.     

Relationship of the regulatory nucleotide site to the catalytic site of the sarcoplasmic reticulum Ca2+-ATPase

The purpose of this study was to probe the regulatory nucleotide site of the Ca2+-ATPase of sarcoplasmic reticulum and to study its relationship with the catalytic nucleotide site. Our approach was to use the nucleotide analogue 2'(3')-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-phosphate (TNP-AMP), which is known to bind the Ca2+-ATPase with high affinity and to undergo a manyfold increase in fluorescence upon enzyme phosphorylation with ATP in the presence of Ca2+. TNP-AMP was shown to bind the regulatory site in that it competitively inhibited (Ki = 0.6 microM) the secondary activation of turnover induced by millimolar ATP, thus providing a high affinity probe for the site. Observation of the high phosphoenzyme-dependent fluorescence upon monomerization of the enzyme without an increase in phosphoenzyme levels showed the regulatory site to be on the same subunit as the catalytic site and excluded an uncovering of "silent" nucleotide sites resulting from dissociation of enzyme subunits. Identical stoichiometric levels of [3H]TNP-AMP binding (4 nmol/mg of protein) to either the free enzyme or the enzyme phosphorylated with 250 microM ATP excluded models of two nucleotide sites per subunit. Finally, transient kinetic experiments in which TNP-AMP was found to block the ADP-induced burst of phosphoenzyme decomposition showed that TNP-AMP was bound to the phosphorylated catalytic site. We conclude that the regulatory nucleotide site is not a separate and distinct site on the Ca2+-ATPase but, rather, results from the nucleotide catalytic site following formation of the phosphorylated enzyme intermediate.     

Does the γ subunit move to an abortive position for ATP hydrolysis when the F1·ADP·Mg complex isomerizes to the inactive F1*·ADP·Mg complex?

F₁-ATPases transiently entrap inhibitory MgADP in a catalytic site during turnover when noncatalytic sites are not saturated with ATP. An initial burst of ATP hydrolysis rapidly decelerates to a slow intermediate rate that gradually accelerates to a final steady-state rate. Transition from the intermediate to the final rate is caused by slow binding of ATP to noncatalytic sites which promotes dissociation of inhibitory MgADP from the affected catalytic site. Evidence from several laboratories suggests that the γ subunit rotates with respect to α/β subunit pairs of F₁-ATPases during ATP hydrolysis. The α₃β₃ and α₃β₃δ subcomplexes of the TF₁-ATPase do not entrap inhibitory MgADP in a catalytic site during turnover, suggesting involvement of the γ subunit in the entrapment process. From these observations, it is proposed that the γ subunit moves into an abortive position for ATP hydrolysis when inhibitory MgADP is entrapped in a catalytic site during ATP hydrolysis.     

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.     

Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location

Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.     

Effects of oligomycin on the partial reactions of the sodium plus potassium-stimulated adenosine triphosphatase

The effects on phosphoenzyme (E-P) formation of ligands which activate Electrophorus (Na,K)-ATPase were investigated in the presence of oligomycin. When the enzyme was allowed to bind oligomycin in the presence of NaCl and MgCl2, subsequent addition of ATP plus KCl produced a monoexponential time course of E-P formation with a rate of 56 s-1, similar to the rate obtained in the uninhibited enzyme phosphorylated by ATP in the absence of KCl. Pi liberation under these conditions was slow and showed no initial burst phase, consistent with the inhibitory effect oligomycin has on the E1-P to E2-P conformational transition. Addition to KCl to a preincubation medium containing oligomycin, NaCl, and MgCl2 had no further effect on E-P formation. However, equilibration with oligomycin, KCl, and MgCl2 prior to the addition of NaCl plus ATP gave a much slower rate of E-P formation (5 s-1) and resulted in an initial rapid release of Pi similar to that found in the uninhibited enzyme. The slow increase in E-P level observed after incubation with oligomycin, KCl, and MgCl2 may be due to secondary formation of an inhibition complex following rapid binding of oligomycin. In contrast to the monophasic behavior which resulted from pre-exposure to NaCl or KCl, preincubation with oligomycin in the presence of MgCl2 plus Tris or Tris alone gave a biphasic pattern of E-P formation in which about 50% of the intermediate accumulated at a rate of 56 s-1 and the remainder at a rate of 5 s-1. In addition, the Pi burst amplitude was reduced, indicating partial inhibition of the enzyme. These results suggest that in the absence of Na+ and K+ only half of the enzyme is inhibited by oligomycin while the remainder undergoes inhibition subsequent to initiation of phosphorylation. Since the oligomycin concentration was saturating, the partial inhibition reflected in the biphasic pattern of E-P formation may be due to half-of-the-sites reactivity in which only half of the subunits bind oligomycin in the absence of monovalent cations.     

Mechanism of inhibition of sarcoplasmic reticulum Ca2(+)-ATPase by active site cross-linking. Impairment of nucleotide binding slows nucleotide-dependent phosphoryl transfer, and loss of active site flexibility stabilizes occluded forms and blocks E2-P formation

Investigation of the properties of Ca2(+)-ATPase of sarcoplasmic reticulum cross-linked at the active site with glutaraldehyde showed that ATP binding affinity and rate of ATP-dependent phosphorylation and Ca2+ occlusion were decreased 2-3 orders of magnitude compared with the native enzyme. Cross-linkage had little effect on or marginally increased the rate of acetyl phosphate- and p-nitrophenyl phosphate-supported Ca2+ occlusion. Ca2+ binding or Ca2(+)-induced changes in tryptophan fluorescence were unaffected. High levels of phosphoenzyme (up to 4 nmol/mg of protein) were obtained, with 2 mol of Ca2+ occluded/mol of E-P. Dephosphorylation and deocclusion occurred together at a slow rate (k = 0.01 s-1) and were stimulated in a monophasic manner up to 20-fold by ADP. Cross-linking inhibited E2-P formation from Pi in 30% (v/v) dimethyl sulfoxide by more than 95%. Induction of turnover of the native ATPase, under conditions designed to yield high steady state levels of E1 approximately P(2Ca), results in a 3-4-fold increase in reactivity of active site residues to glutaraldehyde. The results show that cross-linkage sterically impairs nucleotide binding, changing ATP and ADP into relatively poor substrates, slowing nucleotide-dependent phosphoryl transfer and Ca2+ occlusion and deocclusion. The forward reaction with smaller substrates is unaffected. Another major effect of the cross-link is to inhibit E2-P formation, causing accumulation of E1 approximately P(2Ca) during enzyme turnover and preventing phosphorylation by Pi in the reverse direction. We suggest that occlusion and deocclusion of cations at the transport site of the native enzyme are linked to a two-step cleft closure movement at the active site and that the crosslink stabilizes occluded forms of the pump because it blocks part of this tertiary structural change. The latter could normally be propagated through linking helices to the distal side of the pump to destabilize the cations and open the transport sites to the lumen.     

The calcium ATPase of sarcoplasmic reticulum is inhibited by one Ca2+ ion

Inhibition by calcium of the steady-state turnover of the calcium ATPase from sarcoplasmic reticulum of rabbit muscle follows a Hill slope of 0.8 +/- 0.2 (pH 7.0, 0.1 M KCl, varying [Mg2+] and 2 microM A23187 ionophore). It is concluded that dissociation of the two Ca2+ ions from E-P.Ca2 is sequential and that the inhibition arises from the binding of one Ca2+ to A-P.Ca1.     

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.     

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency

We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence ("superfluorescence") during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.     

Ca2+ binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements.

The Ca2+-binding kinetics of fura-2 and azo-1 were studied using temperature-jump relaxation methods. In 140 mM KCl at 20 degrees C, the association and dissociation rate constants for fura-2 were 6.02 x 10(8) M-1s-1 and 96.7 s-1, respectively. The fura-2 kinetics were insensitive to pH over the range 7.4 to 8.4. Azo-1 was studied in 140 mM KCl, at pH 7.4, at 10 degrees and 20 degrees C. At 10 degrees C, azo-1 exhibited association and dissociation rate constants of 1.43 x 10(8) M-1s-1 and 777.9 s-1, respectively; while at 20 degrees C, the corresponding values were 3.99 x 10(8) M-1s-1 and 1,177 s-1. The kinetic results demonstrate that fura-2 and azo-1 are well suited to monitoring rapid changes in intracellular [Ca2+].