The Ca2+-ATPase partial reactions in cardiac and skeletal sarcoplasmic reticulum. A comparison of transient state kinetic data.

Sumida, Michihiro; Wang, T; Schwartz, Arnold; Younkin, C; Froehlich, Jeffrey P.

doi:10.1016/s0021-9258(19)86059-3

“…A very similar rate constant of 0.8 X 107 M"1 s™1 for ATP binding has been found to be consistent with the time course of enzyme phosphorylation by ATP at pH 6.8 (Sumida et al, 1980), but a larger rate constant of 6 X 107 M'1 s~* was obtained from a simulation for fast kinetics (Fernandez-Belda et al, 1984). Rate constants for ATP dissociation from °E-Ca2-ATP of k_ATP = 100-140 s™1 and for Ca2+ dissociation of fc'_ca = 80 s™1 have been reported previously (Petithory & Jencks, 1986), and a value of k_ca = 55-60 s_1 was measured in this work (see below).…”

Section: Resultssupporting

confidence: 78%

Sequential dissociation of calcium from the calcium adenosinetriphosphatase of sarcoplasmic reticulum and the calcium requirement for its phosphorylation by ATP

Petithory¹,

Jencks²

1988

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The kinetics for dissociation of the stable enzyme-calcium complex of the sarcoplasmic reticulum calcium ATPase, cE.Ca2, were followed by assay with simultaneous addition of [32P]ATP and EGTA, which gives 70% phosphorylation of cE.Ca2 with k = 300 s-1 (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). The binding of ATP to cE.Ca2 is described by kATP = 1.0 X 10(7) M-1 s-1, k-ATP = 120 s-1, and Kdiss = 12 microM; ATP binding is partially rate limiting for phosphorylation at less than 100 microM ATP. The sequential dissociation of Ca2+ from cE.Ca2 is described by k-2 = 55-60 s-1 for the first, "outer" Ca2+, k-1 = 25-30 s-1 for the second, "inner" Ca2+, and K0.5 = 3.4 microM, n = 1.9 (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). Dissociation of the inner Ca2+ is inhibited by external Ca2+, with K0.5 = k-1/k2 = 0.7 microM. This confirms the conclusion that dissociation of the two Ca2+ ions is sequential. The ability of cE.Ca2 to catalyze phosphorylation by ATP disappears in the presence of EGTA with k = 50-55 s-1, the same as k-2 for dissociation of the outer Ca2+ ion. This result, and the absence of the induction period that would occur if both cE.Ca2 and cE.Ca1 were catalytically competent, shows that both Ca2+ ions are required for phosphorylation. This conclusion is confirmed by the stoichiometry of 1.4/0.7 = 2.0 for the ratio of Ca2+ internalized to phosphoenzyme formed after simultaneous addition of ATP and EGTA. Phosphorylation of cE.Ca2 in the presence of 45Ca gives 0.15, not 0.3, 45Ca internalized, which corresponds to exchange of only 1 Ca2+ and is in agreement with this conclusion. The requirements for binding of two Ca2+ for catalytic specificity toward ATP and loss of two Ca2+ from E approximately P.Ca2 for specificity toward water account for the stoichiometry of Ca2+ transport and provide a possible reason for the two steps in the phosphorylation of cE.Ca2.ATP.

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“…Phosphorylation Overshoots. The accumulation of an unphosphorylated form of the enzyme, E-ATP*Ca2, during the steady state can qualitatively explain the overshoot of EP -Ca2 formation that has been observed during phosphorylation under certain experimental conditions (Froehlich & Taylor, 1976;Takisawa & Tonomura, 1978;Sumida et al, 1980). An overshoot is observed when ATP is reacted with enzyme preincubated with calcium, but only if the SRV have not been passively loaded with Ca2+.…”

Section: Discussionmentioning

confidence: 87%

“…Dependence on [Ca2+] of the Reaction of E~P-Ca2 with ADP. The reaction of [32P]E~P-Ca2 with 1.1 mM ADP in the presence of a nonradioactive ATP chase is biphasic, proceeding with a rapid burst of [32P]E~P*Ca2 disappearance followed by a slow phase (Figure 7; Sumida et al, 1980;Froehlich et al, 1980;Pickart & Jencks, 1982; Froehlich & Heller, 1985; Wang, 1986;Fernandez-Belda & Inesi, 1986). The size of the burst is independent of the concentration of Ca2+, but the observed rate constant for phosphoenzyme disappearance in the slow phase increases 2.5-fold when the during the 15 s required to load the solution into the mixer.…”

Section: Resultsmentioning

confidence: 99%

“…Reaction of E~P-Ca2 with ADP. A variety of models has been proposed to explain the biphasic reaction of E~P-Ca2 with ADP, which proceeds with a rapid burst of phosphoenzyme disappearance followed by a slow phase (Sumida et al, 1980;Froehlich et al, 1980; Pickart & Jencks, 1982;Froehlich & Heller, 1985;Wang, 1986; Fernandez-Belda & ). The biphasic reaction has been thought to represent two forms of Ca2+-containing phosphoenzyme (Ej P-Ca2…”

Section: Discussionmentioning

confidence: 99%

“…and E2-P-Ca2) that differ in their sensitivity to ADP, as shown in Scheme I (Sumida et al, 1980;Froehlich et al, 1980). An alternative model is shown in Scheme VI, in which the burst of phosphoenzyme disappearance results from the equilibrium binding of ADP to [32P]E~P-Ca2 followed by rapid phosphoryl transfer to form enzyme-bound [ -32 ] , which dissociates slowly in the rate-limiting step of the slow phase (Pickart & Jencks, 1982;Fernandez-Belda & Inesi, 1986).…”

Section: Discussionmentioning

confidence: 99%

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

Stahl¹,

Jencks²

1987

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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)

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Mechanism of Ca²⁺Transport by Sarcoplasmic Reticulum

Martonosi

¹

,

Beeler

²

1983

Comprehensive Physiology

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The sections in this article are: Structure of Sarcoplasmic Reticulum and Transverse Tubules Structure of Plasmalemma and T Tubules Sarcoplasmic Reticulum Junction Between T Tubules and SR Mechanism of Excitation‐Contraction Coupling Isolation of SR , T Tubules, and Surface Membrane Elements from Skeletal Muscle Separation of Membrane Fractions by Calcium Oxalate or Calcium Phosphate Loading Protein Composition of SR Structure of Ca 2+ ‐Transport ATP ase and Its Disposition in SR Membrane Fragmentation of Ca 2+ ‐ ATP ase With Proteolytic Enzymes Primary Sequence of Ca 2+ ‐Transport ATP ase From Rabbit SR Structure of Proteolipids Structure and Distribution of Calsequestrin and High‐Affinity Ca 2+ ‐Binding Protein in SR Lipid Composition of SR Distribution of Phospholipids in Membrane Bilayer Role of Phospholipids in Atpase Activity and CA 2+ Transport Boundary Lipids and the Problem of Lipid Annulus Rate of ATP Hydrolysis and Physical Properties of the Lipid Phase Mobility of Phospholipids and Ca 2+ ‐Transport ATP ase in SR Mechanism of ATP Hydrolysis and CA 2+ Transport Introduction of Reaction Sequence Ca 2+ Binding to SR Binding of Ca 2+ to Ca 2+ ‐Transport ATP ase Binding of Mg 2+ to Ca 2+ ‐ ATPase Binding of ATP to Ca 2+ ‐ ATPase Binding of Various Substrates to Ca 2+ ‐ ATPase Influence of ATP on Mobility and Reactivity of Protein Side‐Chain Groups Formation of Enzyme‐Substrate Complex Formation and Properties of Phosphoproteins Kinetics of E∼P Formation Relationship Between Enzyme Phosphorylation and Translocation of Calcium Changes in Ca 2+ Affinity of Phosphoenzyme During Ca 2+ Translocation ADP ‐Sensitive and ADP ‐insensitive Phosphoprotein Intermediates Effect of Potassium on ATPase Activity and Ca 2+ Transport Reversal of the CA 2+ Pump Ca 2+ Release Induced by ADP + P i Ca 2+ Gradient‐Dependent Phosphorylation of ATPase by P i Arsenate‐Induced Ca 2+ Release Mechanism of Ca 2+ Release Induced by ADP + P i Ca 2+ Gradient‐Independent Phosphorylation of Ca 2+ ‐ ATPase by P i Role of Ca 2+ ‐Protein Interactions in ATP Synthesis P i HOH Exchange NTP P i Exchange Physical Basis of CA 2+ Translocation Protein‐Protein Interactions in SR and Their Functional Significance Electron Microscopy Fluorescence‐Energy Transfer Electron Spin Resonance Studies ATP ase‐ ATP ase Interactions in Detergent Solutions Chemical Cross‐Linking Effects of Inhibitors on ATPase Activity Possibility of Subunit Heterogeneity Conclusion Permeability of SR Monovalent‐Cation Channels in SR Anion Channels in SR Effect of Membrane Proteins on Permeability of SR Membranes Relationship Between Membrane Potential and Calcium Fluxes Across SR Membrane Probes as Indicators of SR Membrane Potential Influence of SR Membrane Potential on Calcium Permeability Influence of Membrane Potential on Active Calcium Transport Effect of Calcium Uptake on Membrane Potential of SR A Critical Analysis of Experimental Findings on Effects of Ca 2+ Transport on Membrane Potential Effect of Calcium on Optical Response of Positive Cyanine Dyes Response of Negatively Charged Dyes to Calcium Transport by SR Vesicles Membrane Potential of SR In Vivo Effect of Ca 2+ Release on Membrane Potential of SR Transport of CA 2+ by Cardiac SR Kinetic Differences Between SR of Fast‐Twitch and Slow‐Twitch Skeletal Muscles Regulation of CA 2+ Transport by Membrane Phosphorylation Role of Protein Kinase‐Dependent Membrane Phosphorylation in Regulation of Ca 2+ Transport by Skeletal Muscle SR Physiological Significance of Phospholamban Phosphorylation Biosynthesis of SR Studies on SR Development In Vivo Assembly of SR in Cultured Skeletal and Cardiac Muscle Synthesis of Ca 2+ ‐Transport ATPase in Cell‐Free Systems and Its Insertion into the Membrane Synthesis of Calsequestrin Regulation of Synthesis of Ca 2+ ‐Transport ATPase Myogenic Regulation Neural Influence on Concentration of Ca 2+ ‐ ATPase in Muscle Cells

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The Ca2+-ATPase partial reactions in cardiac and skeletal sarcoplasmic reticulum. A comparison of transient state kinetic data.

Cited by 48 publications

References 21 publications

Sequential dissociation of calcium from the calcium adenosinetriphosphatase of sarcoplasmic reticulum and the calcium requirement for its phosphorylation by ATP

Sequential dissociation of calcium from the calcium adenosinetriphosphatase of sarcoplasmic reticulum and the calcium requirement for its phosphorylation by ATP

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

Mechanism of Ca²⁺Transport by Sarcoplasmic Reticulum

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The Ca2+-ATPase partial reactions in cardiac and skeletal sarcoplasmic reticulum. A comparison of transient state kinetic data.

Cited by 48 publications

References 21 publications

Sequential dissociation of calcium from the calcium adenosinetriphosphatase of sarcoplasmic reticulum and the calcium requirement for its phosphorylation by ATP

Sequential dissociation of calcium from the calcium adenosinetriphosphatase of sarcoplasmic reticulum and the calcium requirement for its phosphorylation by ATP

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

Mechanism of Ca2+Transport by Sarcoplasmic Reticulum

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Mechanism of Ca²⁺Transport by Sarcoplasmic Reticulum