Coincidence of H+ binding and Ca2+ dissociation in the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis.

M Yamaguchi,T Kanazawa

doi:10.1016/s0021-9258(18)89156-6

Abstract

H+ and Ca2+ concentration changes in the reaction medium following MgATP addition at pH 6.0 were determined with the partially purified Ca-ATPase from sarcoplasmic reticulum vesicles in the presence of 25-50 microM CaCl2 and 5 mM MgCl2 at 4 degrees C. Previously, we showed a sequential occurrence of H+ binding and H+ dissociation in the Ca-ATPase during ATP hydrolysis and further suggested that the H+ binding takes place inside the vesicles (Yamaguchi, M., and Kanazawa, T. (1984) J. Biol. Chem. 259, 9526-9531). The present results demonstrate that the H+ binding occurred coincidently with Ca2+ dissociation from the enzyme upon conversion of the phosphoenzyme (EP) intermediate from the ADP-sensitive form to the ADP-insensitive form in the catalytic cycle of ATP hydrolysis. As KCl decreased in the medium, the extent of the H+ binding increased almost proportionately with the extent of either the Ca2+ dissociation or the accumulation of ADP-insensitive EP. Both the H+ binding and the Ca2+ dissociation were prevented by a modification of the specific SH group of the enzyme essential for the conversion of ADP-sensitive EP to ADP-insensitive EP. In the late stage of the reaction, H+ dissociation from the enzyme occurred coincidently with Ca2+ binding to the dephosphoenzyme which was formed by EP decomposition. These results are consistent with the possibility that the H+ ejection during the Ca2+ uptake with the intact vesicles previously shown by several investigators takes place through a Ca2+/H+ exchange directly mediated by the membrane-bound Ca-ATPase.

Highlights

Of 25-50 PM CaClzand 6 mM MgClzat 4 OC
Our previous experiment showed a sequential occurrence of H+binding and H+dissociation in the insensitive form in the catalytic cycleof ATP hydrol- Ca-ATPase during ATP hydrolysis [24]
E and EP denote the state of the enzyme which has transport siteswith a high affinity for Caz+and a low affinity for H+

Summary

Motonori Yamaguchiand Tohru KanazawaS

The present results demonstrate that the H+binding occurred coincidently with Ca2+ dissociation from the enzyme upon conversion of the phosphoenzyme (EP) intermediate from the ADP-sensitive form to the ADP-. An important problem to be solved is whether this H+ ejection occurs through a specific mechanism directly involving the Ca-ATPase In this regard, our previous experiment showed a sequential occurrence of H+binding and H+dissociation in the insensitive form in the catalytic cycleof ATP hydrol- Ca-ATPase during ATP hydrolysis [24]. The costs of publication of this article were defrayed in part by the initiated by adding 2.2 pl of mM MgATP This articlemust be hereby containing 36 mMMgC12, adjusted with Tris to pH 6.0) to 2.2 ml of marked “aduertisement” in accordance with 18 U.S.C. Section 1734 the medium containing the enzyme and other reagents as described solely to indicate this fact.

RESULTS

7.5UM HCI '

DISCUSSION