Two-step internalization of calcium from a single E.apprx.P.cntdot.Ca2 species by the calcium ATPase

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Abstract
Phosphorylation by ATP of E.cntdot.*Ca2 (sarcoplasmic reticulum vesicles (SRV) with bound 45Ca2+) during 5-10 ms leads to the occlusion of 2 *Ca2+/EPtot [quench by ethylene glycol bis (.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid (EGTA) alone] in both "empty" (10 .mu.M free Ca2+in) or "loaded" SRV (20-40 mM free Ca2+in). The rate of Ca2+ "internalization" from the occluded E .apprx. P .cntdot. *Ca2 was measured by using an ADP + EGTA quench; a *Ca2+ ion that is not removed by this quench is defined as internalized. In the presence of 20-40 mM unlabeled Ca2+ inside SRV, 1 *Ca2+/EPtot with the same initial rate, but the overall rate constant is kobsd = 17 s-1. The apparent rate constant (kb = 17 s-1) for internalization of the second *Ca2+ is inhibited by [Ca]in, with K0.5 .apprx. 1.3 mM and a Hill coefficient of n = 1.1. These data show that the two Ca2+ ions are internalized sequentially, presumably from separate sequential sites in the channel. [32P]EP .cntdot. Ca2 obtained by rapid mixing of E.cntdot.Ca2 with [.gamma.-32P]ATP and EGTA disappears in a biphasic time course with a lag corresponding to .apprx. 34 s-1, followed by EP* decay with a rate constant of .apprx. 17 s-1. This shows that both Ca2+ ions must be internalized before the enzyme changes its specificity for catalysis of posphoryl transfer to water instead of to ADP. Increasing the concentration of ATP from 0.25 to 3 mM accelerates the rate of 45Ca2+ internalization from 34 to 69 s-1 for the first Ca2+ and from 17 to 34 s-1 for the second Ca2+. High [ATP] also accelerates both phases of [32P]EP .cntdot. Ca2 disappearance by the same factor. The data are consistent with a single form of ADP-sensitive E .apprx. P .cntdot. Ca2 that sequentially internalizes two ions. The intravesicular volume was estimated to be 2.0 .mu.L/mg, so that one turnover of the enzyme gives 4 mM internal [Ca2+].

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