The effect of di- and trivalent cations on the phosphorylation of the Ca2+-ATPase in sarcoplasmic reticulum vesicles

Domonkos, J; Heiner, L; Vargha, M

doi:10.1016/0005-2736(85)90061-6

Cited by 5 publications

(4 citation statements)

References 41 publications

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“…The decrease in the amount of EP formation from Mg-ATP when the enzyme is preincubated with both La3+ and Ca2+ (Table I) is consistent with binding of La3+ to the exterior calcium transport sites (Chevallier & Butow, 1971), which inhibits formation of the phosphoenzyme. Increasing the ratio of La3+ to Ca2+ in the preincubation solution decreases the amount of EP formation at 2 s. This shows both that La3+ can compete with the binding of Ca2+ (Domonokos et al, 1985) and that La3"1" is not a congener of Ca2+ for the CaATPase reaction. The observed decreases in the amount of EP formation shown in Table I are consistent with the model of eq 7 with KCi = 4.0 X 10"12 M2 and K0 5 in the range of…”

Section: Discussionmentioning

confidence: 93%

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

Hanel

Jencks²

1990

Biochemistry

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The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE.Ca2) is phosphorylated upon the addition of 0.25 mM LaCl3 and 0.3 mM [gamma-32P]ATP with an observed rate constant of 6.5 s-1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 degrees C). La.ATP binds to cE.Ca2 with a rate constant of 5 X 10(6) M-1 s-1, while ATP, Ca2+, and La3+ dissociate from cE.Ca2.La.ATP at less than or equal to 1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La.ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP.ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (alpha) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.34 and 65 s-1, respectively, at saturating ADP (KADPS = 0.22 mM). The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE.Ca2 by La.ATP at 6.5 s-1 and the kinetics for the reaction of EP with ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE.Ca2.La.ATP to the form of the enzyme that is activated for phosphoryl transfer, aE.Ca2.La.ATP, at 6.5 s-1; this is much slower than the analogous conformational change at 220 s-1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE.Ca2.La.ATP to cE.Ca2.La.ATP is 170 s-1. ATP does not dissociate measurably from aE.Ca2.La.ATP. Labeled EP formed from cE.Ca2 and La.ATP with leaky vesicles undergoes hydrolysis at 0.06 s-1. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg.ATP and La.ATP; however, the strong binding of La.ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La.ATP. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE-Ca2 is mixed with 0.3 mM ATP and 1.0 mM Cacl2, the phosphoenzyme is formed with an observed rate constant of 3 s-1. The phosphoenzyme formed from Ca.ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s-1; there may be a small burst (alpha less than or equal to 0.05).

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Section: Discussionmentioning

confidence: 93%

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

Hanel

Jencks²

1990

Biochemistry

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“…The overall rate of catalysis (ATPase activity) is slowed with 0006-2960/93/0432-6632S04.00/0 © 1993 American Chemical Society Scheme II Ca2+, La3+, Cr3+, or Co2+ as the catalytic ion (Vianna, 1975;Garrahan et al, 1976; Yamada & Ikemoto, 1980;Wakabayashi & Shigekawa, 1984; Yamada et al, 1986; Vilsen & Andersen, 1987; Fujimori & Jencks, 1990; Hanel & Jencks, 1990), whereas Mn2+ or Zn2+ allow turnover of ATP at a rate similar to that observed with Mg2+ (Yamada & Ikemoto, 1980;Chiesi & Inesi, 1981;Henao & Gutierrez-Merino, 1989;Ogurusuetal., 1991). The rates of phosphoenzyme formation from ATP and its hydrolytic breakdown are decreased to a similar extent if Mg2+ is replaced by Ca2+ or La3+ as the catalytic ion (Garrahan et al, 1976;Domonkos et al, 1985;Fujimori & Jencks, 1990).…”

Section: Scheme Imentioning

confidence: 99%

The binding of ATP and magnesium to the calcium adenosinetriphosphatase of sarcoplasmic reticulum follows a random mechanism

Reinstein

Jencks²

1993

Biochemistry

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The enzyme form of the calcium adenosinetriphosphatase of sarcoplasmic reticulum (CaATPase) that is stable in the presence of calcium, cE.Ca2, has a binding site for the catalytic Mg2+ ion with a dissociation constant of 0.94 +/- 0.15 mM at 25 degrees C, pH 7.0, and 100 mM KCl. This is approximately 10 times smaller than that reported for the free enzyme, E, (8.8 mM) under similar conditions [Punzengruber, C., Prager, R., Kolassa, N., Winkler, F., & Suko, J. (1978) Eur. J. Biochem. 92, 349-359]. This difference shows that the sites for the catalytic and the transported ions interact in the absence of ATP. The addition of ATP and EDTA to enzyme that had been incubated with Ca2+ and Mg2+ resulted in the formation of 61% phosphoenzyme. The addition of unlabeled ATP and Mg2+ to enzyme that had been incubated with 3.5 microM free Ca2+ and labeled ATP gave 39% labeled phosphoenzyme. This shows that the binding of ATP and Mg2+ to cE.Ca2 follows a random mechanism. The rate constants for dissociation of ATP and Mg2+ from cE.Ca2.ATP.Mg are different: kdiss(ATP) = 120 s-1 and kdiss(Mg2+) = 60 s-1. This shows that Mg2+ and ATP can bind and dissociate independently; they do not have to associate or dissociate from cE as a Mg.ATP complex. Calcium-free enzyme binds metal-free ATP at the active site with a dissociation constant of 44 +/- 4 microM, kdiss = 130 +/- 7 s-1, and a calculated association rate constant of 3 x 10(6) M-1 s-1. Calcium-free enzyme that was incubated with [gamma-32P]ATP gave 38% labeled phosphoenzyme when chased with unlabeled ATP, Mg2+, and Ca2+. An increase of the Mg2+ concentration did not increase the amount of E32P formed. This shows that the binding of Mg2+ and ATP to free E also follows a random mechanism. The Mg2+ ion is not buried under ATP, and ATP is not under a Mg2+ ion. Incubation of free E with Mg2+ and ATP causes a conformational change that activates the enzyme for phosphorylation and decreases the rate constant for the dissociation of ATP from kdiss = 120 s-1 to kdiss = 47 s-1.

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“…Step-function electron density model for the SR membrane profile for T = 150C and [Mg2"] 100 AM. Five steps (1)(2)(3)(4)(5) are assigned to the membrane extravesicular surface region extending from beyond the lipid polar head groups facing the extravesicular space to the edge of the unit cell.…”

Section: Modeling the Membrane Electron Density Profilesmentioning

confidence: 99%

“…The effect of temperature and Mg2`concentration on the kinetics of Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles has been extensively studied (1)(2)(3)(4). Previous work in our laboratory established a relation between the temperature dependence of Ca2`uptake by SR vesicles and variations in the electron density profile structure of the membrane (5).…”

Section: Introductionmentioning

confidence: 99%

Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane

Asturias

Blasie

1989

Biophysical Journal

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Direct measurements of phosphorylation of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) have shown that the lifetime of the first phosphorylated intermediate in the Ca2+ transport cycle, E1 approximately P, increases with decreasing [Mg2+] (Dupont, Y. 1980. Eur. J. Biochem. 109:231-238). Previous x-ray diffraction work (Pascolini, D., and J.K. Blasie. 1988. Biophys. J. 54:669-678) under high [Mg2+] conditions (25 mM) indicated that changes in the profile structure of the SR membrane could be responsible for the low-temperature transient trapping of E1 approximately P that occurs at temperatures below 2-3 degrees C, the upper characteristic temperature th for lipid lateral phase separation in the membrane. We now present results of our study of the Ca2+ uptake kinetics and of the structure of the SR membrane at low [Mg2+] (less than or equal to 100 microM). Our results show a slowing in the kinetics of both phases of the Ca2+ uptake process and an increase in the duration of the plateau of the fast phase before the onset of the slow phase, indicating an increase in the lifetime (transient trapping) of E1 approximately P. Calcium uptake kinetics at low [Mg2+] and moderately low temperature (approximately 0 degree C) are similar to those observed at much lower temperatures (approximately -10 degrees C) at high [Mg2+]. The temperature-induced structural changes that we observed at low [Mg2+] are much more pronounced than those found to occur at higher [Mg2+]. Also, at the lower [Mg2+] the upper characteristic temperature th for lipid lateral phase separation was found to be higher, at approximately 8-10 degrees C. Our studies indicate that both temperature and [Mg2+] affect the structure and the functionality (as measured by changes in the kinetics of Ca2+ uptake) of the SR membrane. Membrane lipid phase behavior and changes in the Ca2+ ATPase profile structure seem to be related, and we have found that structural changes are responsible for the slowing of the kinetics of the fast phase of Ca2+ uptake, and could also mediate the effect that [Mg2+] has on E1 approximately P lifetime.

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The effect of di- and trivalent cations on the phosphorylation of the Ca2+-ATPase in sarcoplasmic reticulum vesicles

Cited by 5 publications

References 41 publications

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

The binding of ATP and magnesium to the calcium adenosinetriphosphatase of sarcoplasmic reticulum follows a random mechanism

Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane

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