Two-step Internalization of Ca²⁺From A Single E~P*ca₂species By The Ca²⁺-atpase^f

Phosphorylation by ATP of E-*Ca₂(sarcoplasmic reticulum vesicles (SRV) with bound⁴⁵Ca²⁺) during 5–10 ms leads to the occlusion of 2 *Ca²⁺/EP_tot[quench by ethylene glycol bis(/3-aminoethyl ether)-iV,yV,A^r/,A^r-tetraacetic acid (EGTA) alone] in both “empty” (10 pM free Ca²⁺_in) or “loaded” SRV (20–40 mM free Ca²⁺_in). The rate of Ca²⁺“internalization” from the occluded E~PCa₂was 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⁺/EP_totis internalized from⁴⁵Ca-labeled E~PCa₂with a first-order rate constant of k_x= 34 s¹. Empty SRV take up 2 *Ca²⁺/EP_totwith the same initial rate, but the overall rate constant is fc_obsd= 17 s-¹. The apparent rate constant (k_h= 17 s”¹) for internalization of the second *Ca²⁺is inhibited by [Ca]_in, with K_os~ 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.Ca₂obtained by rapid mixing of E.Ca₂with[y- ³²P]ATP and EGTA disappears in a biphasic time course with a lag corresponding to ~34 s”¹, followed by EP* decay with a rate constant of ~ 17 s¹. This shows that both Ca²⁺ions must be internalized before the enzyme changes its specificity for catalysis of phosphoryl transfer to water instead of to ADP. Increasing the concentration of ATP from 0.25 to 3 mM accelerates the rate of⁴⁵Ca²⁺internalization from 34 to 69 s^-1for the first Ca²⁺and from 17 to 34 s^_1for the second Ca²⁺. High [ATP] also accelerates both phases of [³²P]EP-Ca₂disappearance by the same factor. The data are consistent with a single form of ADP-sensitive E~P•Ca₂that sequentially internalizes two ions. The intravesicular volume was estimated to be 2.0 ^L/mg, so that one turnover of the enzyme gives 4 mM internal [Ca²⁺].