Conformational changes in the vicinity of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle

In the previous experiment (Suzuki, H., Obara, M., Kuwayama, H., and Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456), the Ca2+-ATPase of sarcoplasmic reticulum vesicles was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The main labeled site was Cys674. A large monophasic fluorescence drop occurred upon ATP binding to the catalytic site of the Ca2+-activated enzyme in the presence of K+. The present results show that this fluorescence drop is biphasic in the absence of K+. The first and rapid phase of this drop accounts for most of the fluorescence drop. This phase reflects a conformational change in the enzyme.ATP complex. The second and slow phase, being much smaller than the first phase, coincides with phosphoenzyme (EP) isomerization from the ADP-sensitive form to the ADP-insensitive form. This phase disappears when accumulation of ADP-insensitive EP is inhibited by K+ or when EP isomerization is prevented by the N-ethylmaleimide treatment. These results show that this phase reflects a conformational change upon EP isomerization. When free Ca2+ is chelated after EP formation from ATP, the fluorescence intensity is restored to the initial level without Ca2+. This restoration coincides with EP decomposition. This suggests that the fluorescence restoration reflects a conformational change upon hydrolysis of ADP-insensitive EP. This probability is supported by the concurrent occurrence of the Pi-induced fluorescence drop and EP formation from Pi. The results demonstrate that the fluorescence drop upon ATP binding is predominant in the fluorescence change throughout the catalytic cycle.     

Characterization of the substrate-induced conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2(+)-ATPase by using different kinds of substrate

Cys-674 of the sarcoplasmic reticulum Ca2(+)-ATPase was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and changes in the fluorescence intensity upon addition of seven kinds of substrate were followed by the stopped-flow method. The steady-state fluorescence intensity and anisotropy were also determined. When Ca2+ was present, the fluorescence intensity and anisotropy decreased greatly upon addition of any substrate used. The observed affinity for each substrate agreed with the previously observed affinity of the catalytic site. The fluorescence drop induced by the adenine nucleotides, ATP and adenosine 5'-(beta, gamma-methylene)triphosphate (a nonhydrolyzable ATP analog), was much faster than that induced by other substrates. The ATP-induced fluorescence drop preceded phosphoenzyme formation when the ATP concentration was high, but the fluorescence drop coincided with phosphoenzyme formation when it was slowed by reducing ATP concentrations. The fluorescence drop induced by ITP or acetyl phosphate was slow even at high concentrations of the substrate, and it coincided with phosphoenzyme formation. When Ca2+ was absent, the fluorescence intensity and anisotropy decreased only slightly upon addition of any substrate other than the adenine nucleotides. They decreased substantially upon addition of the adenine nucleotides, but the kinetics of this fluorescence drop were quite different from that of the fluorescence drop induced by any substrate in the presence of Ca2+. These results show that the conformational change, which makes the bound label less constrained, is induced by substrate binding to the catalytic site of the Ca2(+)-activated enzyme. This change precedes phosphoenzyme formation in the catalytic cycle and is greatly accelerated by the adenine moiety of the substrate.     

Changes in the steady-state fluorescence anisotropy of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle

Cys674 of the sarcoplasmic reticulum Ca2+-ATPase was selectively labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and the steady-state fluorescence anisotropy of this label and its total fluorescence intensity were followed throughout the catalytic cycle. At 25 degrees C, the anisotropy and the total fluorescence intensity increased by 2.1 and 9.4%, respectively, upon Ca2+ binding to the high affinity sites. Upon subsequent ATP binding to the catalytic site, the anisotropy and the total fluorescence intensity decreased by 6.8 and 23.9%, respectively. These drops likely occurred in the enzyme.ATP complex. The extents of changes upon additions of Ca2+ and ATP in the anisotropy, but not in the total fluorescence intensity, were greatly reduced by lowering the temperature. Slight drops in the anisotropy and the total fluorescence intensity occurred upon conversion of phosphoenzyme (EP) from the ADP-sensitive form to the ADP-insensitive form. The anisotropy and the total fluorescence intensity returned to the initial level when EP was hydrolyzed. Mg2+-dependent Pi-induced drops in the anisotropy and the total fluorescence intensity occurred coincidently with EP formation from Pi. These demonstrate that the ATP-induced drops in the anisotropy and the total fluorescence intensity are predominant throughout the catalytic cycle. Most probably, the changes in the anisotropy are due to changes in the rotational diffusion of the label. These findings indicate that ATP binding to the catalytic site induces a relaxed conformation in the microenvironment of the label bound to Cys674.     

Binding of two Sr2+ ions changes the chemical specificities for phosphorylation of the sarcoplasmic reticulum calcium ATPase through a stepwise mechanism.

The sequential binding of Sr2+ and Ca2+ to the cytoplasmic transport sites of the sarcoplasmic reticulum calcium ATPase allows the formation of two different mixed complexes: cE.Sr.Ca, with Sr2+ bound to the "inner" site and Ca2+ bound to the "outer" site, and cE. Ca.Sr, with Ca2+ bound to the inner site and Sr2+ bound to the outer site (pH 7.0, 25 degrees C, 10 mM MgCl2, 100 mM KCl). Both cE.Sr.45Ca and cE.45Ca.Sr react with ATP to internalize one 45Ca/phosphoenzyme. The value of K0.5 = 83 microM Sr2+ for activation of the enzyme for phosphorylation by ATP is much larger than K0.5 = 28 microM Sr2+ for inhibition of phosphoenzyme formation from inorganic phosphate (eta H = 1.0-1.3). These results are consistent with the sequential binding of two strontium ions with negative cooperativity and dissociation constants of KSr1 = 35 microM and KSr2 = 55 microM. The species cE.Sr2 and cE.Ca2 react rapidly with ATP but not inorganic phosphate. However, enzyme with one strontium bound, cE.Sr, does not react with either inorganic phosphate or ATP. Therefore, the conformational changes in the enzyme that alter the chemical specificity for phosphorylation by ATP and by inorganic phosphate are different. This requires the existence of at least three forms of the unphosphorylated enzyme with three different chemical specificities for catalysis.     

Inhibition by A23187 of conformational changes involved in the Ca(2+)-induced activation of sarcoplasmic reticulum Ca(2+)-ATPase.

We previously showed that A23187 in high ionophore/protein ratios almost completely inhibits the sarcoplasmic reticulum Ca(2+)-ATPase [Hara, H. & Kanazawa, T. (1986) J. Biol. Chem. 261, 16584-16590]. In an attempt to obtain information on the mechanism of this inhibition, the effects of A23187 on conformational changes involved in the Ca(2+)-induced activation of the enzyme were investigated. The purified enzyme from sarcoplasmic reticulum of rabbit skeletal muscle as well as the purified enzyme labeled with fluorescein 5-isothiocyanate (FITC) were preincubated with A23187 in the absence of Ca2+ at pH 7.0 and 0 degrees C for 45 min. The activation of the enzyme following addition of CaCl2 was assessed by determining the capacity for rapid formation of phosphoenzyme from ATP. This activation was strongly inhibited by the preincubation with A23187. This indicates that the previously observed inhibition of the Ca(2+)-ATPase is mostly due to hindrance of the Ca(2+)-induced activation of the enzyme. In the control, in which the FITC-labeled enzyme was preincubated without A23187, the fluorescence intensity of the bound FITC decreased in a biphasic manner upon addition of CaCl2. The first rapid phase of this fluorescence drop was unaffected by A23187, whereas its second slow phase was almost completely inhibited by this drug. These results show that the Ca(2+)-dependent conformational change is biphasic and that the second slow phase (but not the first rapid phase) of this conformational change is inhibited by A23187. This suggests that the observed inhibition of Ca2+ activation is attributed to hindrance of the second slow phase of the Ca(2+)-dependent conformational change.     

Conformational basis of the phospholipid requirement for the activity of SR Ca(2+)-ATPase

The delipidated sarcoplasmic reticulum (SR) Ca(2+)-ATPase was reconstituted into proteoliposomes containing different phospholipids. The result demonstrated the necessity of phosphatidylcholine (PC) for optimal ATPase activity and phosphatidylethanolamine (PE) for the optimal calcium transport activity. Fluorescence intensity of Fluorescein 5-isothiocyanate (FITC)-labeled enzyme at Lys515 as well as the measurement of the distance between 5-((2-[(iodoacetyl) amino] ethyl) amino)naphthalene-1-sulphonic acid (IAEDANS) label sites (Cys674/670) and Pr3+ demonstrated a conformational change of cytoplasmic domain, consequently, leading to the variation of the enzyme function with the proteoliposomes composition. Both the intrinsic fluorescence of Trp and its dynamic quenching by HB decreased with increasing PE content, revealing the conformational change of transmembrane domain. Time-resolved fluorescence study characterized three classes of Trp residues, which showed distinctive variation with the change in phospholipid composition. The phospholipid headgroup size caused the conformational change of SR Ca(2+)-ATPase, subsequent the ATPase activity and Ca2+ uptake.     

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.     

Mutations of either or both Cys876 and Cys888 residues of sarcoplasmic reticulum Ca2+-ATPase result in a complete loss of Ca2+ transport activity without a loss of Ca2+-dependent ATPase activity. Role of the CYS876-CYS888 disulfide bond.

Disulfide-containing peptides in pepsin digest of sarcoplasmic reticulum vesicles were identified by using a fluorogenic thiol-specific reagent 4-fluoro-7-sulfamoylbenzofurazan and a reductant tributylphosphine. Sequencing of the purified peptides revealed the presence of a Cys(876)-Cys(888) disulfide bond on the luminal loop connecting the 7th and 8th transmembrane helices (loop 7-8) of the Ca(2+)-ATPase (SERCA1a). We substituted either or both of these cysteine residues with alanine and made three mutants (C876A, C888A, C876A/C888A), in which the disulfide bond is disrupted. The mutants and the wild type were expressed in COS-1 cells, and functional analysis was performed with the microsomes isolated from the cells. Electrophoresis performed under reducing and non-reducing conditions confirmed the presence of Cys(876)-Cys(888) disulfide bond in the expressed wild type. All the three mutants possessed high Ca(2+)-ATPase activity. In contrast, no Ca(2+) transport activity was detected with these mutants. These mutants formed almost the same amount of phosphoenzyme intermediate as the wild type from ATP and from P(i). Detailed kinetic analysis showed that the three mutants hydrolyze ATP in the mechanism well accepted for the Ca(2+)-ATPase; activation of the catalytic site upon high affinity Ca(2+) binding, formation of ADP-sensitive phosphoenzyme, subsequent rate-limiting transition to ADP-insensitive phosphoenzyme, and hydrolysis of the latter phosphoenzyme. It is likely that the pathway for delivery of Ca(2+) from the binding sites into the lumen of vesicles is disrupted by disruption of the Cys(876)-Cys(888) disulfide bond, and therefore that the loop 7-8 having the disulfide bond is important for formation of the proper structure of the Ca(2+) pathway.     

Acceleration of the rate of fluorescence decrease by high concentrations of ATP under the condition of accumulation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase

Addition of up to 300 microM ATP in the presence of 2 M NaCl with MgCl2 to pig kidney Na+,K+-ATPase treated with N-[p-(2-benzimidazolyl)phenyl]maleimide seemed to be insufficient to saturate the rate of the fluorescence decrease. However, both the extent of the decrease and the amount of phosphoenzyme at a steady state were saturated below 20 microM ATP. Addition of Mg2+ with Na+ to the enzyme preincubated with 20 to 600 microM ATP gave nearly the same rate constant, which was below 50% of that obtained by adding 300 microM ATP to the Na+-form enzyme in the presence of Mg2+. High concentrations of ATP affected neither the rate of light-scattering change (Taniguchi, K. et al. (1986) J. Biol. Chem. 261, 3272-3281) after ADP-sensitive phosphoenzyme formation (E1P) nor that of the breakdown of E1P. A stoichiometric amount of [32P]Pi was liberated from [32P]E1P. The data suggested that ATP did not bind to E1P in such a way as to increase the extent of phosphorylation further or to accelerate dephosphorylation. The data also suggested that the reason for the large difference in the apparent affinity of ATP as evaluated from the rate and the extent of fluorescence change is the large dissociation constant for ATP of a Michaelis complex.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.     

A fluorescence probe study of the phosphorylation reaction of the calcium ATPase of sarcoplasmic reticulum

The fluorescent thiol reagent N-(1-anilinonaphthyl-4)maleimide (ANM) reacts covalently with the Ca2+ ATPase moiety of fragmented sarcoplasmic reticulum in two phases as determined by the increase of fluorescence intensity and optical density at 350 nm. In the rapid phase, 5.5 nmol of ANM reacts with 1 mg of fragmented sarcoplasmic reticulum protein. Assuming that 55% of the total membrane protein is the Ca2+ ATPase, this is equivalent to 1 mol of SH/10(5) g of ATPase, designated as SH1-ANM. ANM reacts with the second SH (SH2-ANM) at a much slower rate. Reaction of ANM with both SH1-ANM and SH2-ANM produces no inhibition of phosphoenzyme (EP) formation. Upon addition of Mg . ATP in the micromolar range, at [Ca2+] = 1 microM there is an increase in the fluorescence intensity of ANM attached to SH2-ANM, while the ANM attached to SH1-ANM does not respond to Mg . ATP. Under conditions in which there is no EP formation, there is no fluorescence change. Furthermore, the enhancement of ANM fluorescence produced by Mg . ATP is reversed by ADP as it reacts with EP to form ATP. Thus, it appears that the Mg . ATP-induced fluorescence increase reflects changes of enzyme conformation produced by EP formation.     

Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase

Fluorescein isothiocyanate was used to covalently label the gastric (H+ + K+)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated p-nitrophenylphosphatase activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of pI 5.6 was also noted. Fluorescence was quenched by K+, Rb+ and Tl+ in a dose-dependent manner, and the K0.5 values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg2+-dependent fluorescent quench. Ca2+ reversed the K+-dependent loss of fluorescence and inhibited it when added prior to K+. This may relate to the slow phosphorylation in the presence of ATP found when Ca2+ was substituted for Mg2+ and the absence of K+-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K+ binding to the enzyme.     

Reactions of the sarcoplasmic reticulum calcium adenosinetriphosphatase with adenosine 5'-triphosphate and Ca2+ that are not satisfactorily described by an E1-E2 model

Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with kb = 70 +/- 7 s-1 after free enzyme was mixed with saturating ATP and 50 microM Ca2+; this is one-third the rate constant of 220 s-1 for phosphorylation of enzyme preincubated with calcium, cE.Ca2, after being mixed with ATP under the same conditions (pH 7.0, Ca2+-loaded vesicles, 100 mM KCl, 5 mM Mg2+, 25 degrees C). Phosphorylation of E with ATP and Ca2+ in the presence of 0.25 mM ADP gives approximately 50% E approximately P.Ca2 with kobsd = 77 s-1, not the sum of the forward and reverse rate constants, kobsd = kf + kr = 140 s-1, that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) kb represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of cE.Ca2. The absence of a lag for phosphorylation of E with saturating ATP and Ca2+ indicates that all other steps, including the binding of Ca2+ ions and phosphoryl transfer, have rate constants of greater than 500 s-1. Chase experiments with unlabeled ATP or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) show that the rate constants for dissociation of [gamma-32P]ATP and Ca2+ are comparable to kb. Dissociation of ATP occurs at 47 s-1 from E.ATP.Ca2+ and at 24 s-1 from E.ATP. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 microM ATP and 50 microM Ca2+ to enzyme. This shows that Ca2+ binds rapidly to the free enzyme, from outside the vesicle, before the conformational change (kb). The fraction of Ca2+-free E.[gamma-32P]ATP that is trapped to give labeled phosphoenzyme after the addition of Ca2+ and a chase of unlabeled ATP is half-maximal at 6.8 microM Ca2+, with a Hill slope of n = 1.8. The calculated dissociation constant for Ca2+ from E.ATP.Ca2 is approximately 2.2 X 10(-10) M2 (K0.5 = 15 microM). The rate constant for the slow phase of the biphasic reaction of E approximately P.Ca2 with 1.1 mM ADP increases 2.5-fold when [Ca2+] is decreased from 50 microM to 10 nM, with half-maximal increase at 1.7 microM Ca2+. This shows that Ca2+ is dissociating from a different species, aE.ATP.Ca2, that is active for catalysis of phosphoryl transfer, has a high affinity for Ca2+, and dissociates Ca2+ with k less than or equal to 45 s-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme.

The internalization of 45Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25 degrees C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS.Tris, pH 7.0). Intact vesicles containing either 10-20 microM or 20 mM Ca2+ were preincubated with 45Ca for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of Etot with the rate constant k = 300 s-1. The two 45Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and 45Ca with excess 40Ca, or by mixing the enzyme and 40Ca with 45Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with kc = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater than or equal to 10(5) s-1) than the forward and reverse reactions for the conformational change (k-c = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (kmix greater than or equal to 10(5) s-1) before dissociating. A Hill slope of nH = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.     

5-Iodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover

The fluorescence of (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein was studied under turnover conditions. At 4 degrees C the hydrolysis of ATP is slowed sufficiently to permit study of the effects of Na+, K+, and ATP on the steady-state intermediates. With Na+ and Mg2+ (Na-ATPase conditions), addition of ATP produces a 7% drop in signal that reverts back to the initial, high fluorescence after a steady state of several minutes. K-sensitive phosphoenzyme is formed under these conditions, indicating that the fluorescence signal during the steady state is associated with E2P. Under (Na,K)-ATPase conditions (Na+, K+, Mg2+), micromolar ATP produces a steady-state signal that is 25% lower than the initial fluorescence, with no detectable phosphoenzyme formed. This low-fluorescence intermediate, which is also formed by adding K+ to enzyme in the Na-ATPase steady state described above, resembles the state produced by adding K+ directly to enzyme under equilibrium conditions, i.e. E2K. The K0.5(K+) for the fluorescence decrease and for keeping the enzyme dephosphorylated are nearly identical, indicating that the fluorescence change accompanies K+-dependent dephosphorylation. High ATP increases the steady-state fluorescence during the (Na,K)-ATPase reaction; while oligomycin produces still another steady-state fluorescent intermediate. These last two intermediates may be associated with the formation of E2P and E1P, respectively.     

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.     

The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues

Since Na+,K+-ATPase (EC 3.6.1.3) of pig kidney modified with a fluorescent sulfhydryl reagent, N-[p-(2-benzimidazolyl) phenyl]maleimide, at Cys-964 of the alpha-chain showed ATP-dependent, reversible, and dynamic fluorescence changes (Nagai, M., Taniguchi, K., Kangawa, K., Matsuo, S., Nakamura, S., and Iida, S. (1986) J. Biol. Chem. 261, 13197-13202), we studied the conformational change during Na+,K+-ATPase reaction using the modified enzyme. The addition of K+ to the enzyme increased the fluorescence intensity to 2% in the presence of 160 mM Na+ and 3 mM Mg2+ (K0.5 = 16.4 mM). Addition of low concentrations of ATP immediately increased the intensity to 3.2% (K0.5 less than 0.1 microM) to accumulate fully K+-bound enzyme in the presence of 43 mM K+ with Na+ and Mg2+, but further addition of higher concentrations of ATP diminished the increase (K0.5 = 120 microM). After exhaustion of ATP, the fluorescence intensity decreased to -0.4% (K0.5 = 0.3 microM) and -2% (K0.5 = 20 microM), respectively, in the presence of low and high concentrations of ADP produced from ATP. High concentrations of ATP accelerated Na+,K+-ATPase activity with a simultaneous increase in the amount of ADP-sensitive phosphoenzyme irrespective of the modification. Adenylyl imidodiphosphate and ADP accelerated Na+,K+-ATPase activity in the presence of 2.7 microM ATP by decreasing the extent of the fluorescence without affecting the amount of phosphoenzyme, irrespective of the modification. These data suggest that Na+,K+-ATPase activity was accelerated due to the acceleration of the breakdown of K+-bound enzyme by high concentrations of ATP and ATP analogues.     

Phosphorylation of the calcium adenosinetriphosphatase of sarcoplasmic reticulum: rate-limiting conformational change followed by rapid phosphoryl transfer

The calcium adenosinetriphosphatase of sarcoplasmic reticulum, preincubated with Ca2+ on the vesicle exterior (cE X Ca2), reacts with 0.3-0.5 mM Mg X ATP to form covalent phosphoenzyme (E approximately P X Ca2) with an observed rate constant of 220 s-1 (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4, 23 microM free external Ca2+, intact SR vesicles passively loaded with 20 mM Ca2+). If the phosphoryl-transfer step were rate-limiting, with kf = 220 s-1, the approach to equilibrium in the presence of ADP, to give 50% EP and kf = kr, would follow kobsd = kf + kr = 440 s-1. The reaction of cE X Ca2 with 0.8-1.2 mM ATP plus 0.25 mM ADP proceeds to 50% completion with kobsd = 270 s-1. This result shows that phosphoryl transfer from bound ATP to the enzyme is not the rate-limiting step for phosphoenzyme formation from cE X Ca2. The result is consistent with a rate-limiting conformational change of the cE X Ca2 X ATP intermediate followed by rapid (greater than or equal to 1000 s-1) phosphoryl transfer. Calcium dissociates from cE X Ca2 X ATP with kobsd = 80 s-1 and ATP dissociates with kobsd = 120 s-1 when cE X Ca2 X ATP is formed by the addition of ATP to cE X Ca2. However, when E X Ca2 X ATP is formed in the reverse direction, from the reaction of E approximately P X Ca2 and ADP, Ca2+ dissociates with kobsd = 45 s-1 and ATP dissociates with kobsd = 35 s-1. This shows that different E X Ca2 X ATP intermediates are generated in the forward and reverse directions, which are interconverted by a conformational change.