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

Daniel Khananshvili, William P. Jencks

Research output: Contribution to journalArticlepeer-review

Abstract

Phosphorylation by ATP of E-*Ca2(sarcoplasmic reticulum vesicles (SRV) with bound45Ca2+) during 5–10 ms leads to the occlusion of 2 *Ca2+/EPtot[quench by ethylene glycol bis(/3-aminoethyl ether)-iV,yV,Ar/,Ar-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+/EPtotis internalized from45Ca-labeled E~PCa2with a first-order rate constant of kx= 34 s1. Empty SRV take up 2 *Ca2+/EPtotwith the same initial rate, but the overall rate constant is fcobsd= 17 s-1. The apparent rate constant (kh= 17 s”1) for internalization of the second *Ca2+is inhibited by [Ca]in, with Kos~ 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+].

Original languageEnglish
Pages (from-to)2943-2952
Number of pages10
JournalBiochemistry
Volume27
Issue number8
DOIs
StatePublished - 1 Apr 1988
Externally publishedYes

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