ACTIVATION OF SARCOPLASMIC-RETICULUM CA2+-ATPASE BY MN2+ - A MN2+ BINDING STUDY

We used Mn2+ as an analogue for Mg2+ to examine the minimum requirement of divalent cations for the rapid turnover of the sarcoplasmic reticulum ATPase. We measured the binding of Ca2+ and Mn2+ to the purified Ca2+-ATPase during steady-state hydrolysis of MnATP at 2-degrees-C and pH 7. In the presence of 20-mu-M Ca2+, Mn2+ was as effective as Mg2+ in stimulating ATPase activity and the maximal activation of ATP hydrolysis was observed at 0.1 mM MnCl2. Under these conditions, 2 mol of Ca2+ were bound per mol of the ADP-sensitive phosphoenzyme, whereas no Ca2+ was bound to the ADP-insensitive phosphoenzyme. On the other hand, the stoichiometry for ATP-dependent binding of Mn2+ to these intermediates was about 1. We found that Mn2+ remained bound to the ADP-insensitive phosphoenzyme even in the presence of added chelator. In the absence of ATP, we detected a low level of Mn2+ binding, which reached 0.4 mol per mol of the phosphorylation site at 0.1 mM free Mn2+. We present evidence that this extra Mn2+ binding did not affect the rate of decomposition of the ADP-sensitive phosphoenzyme, which was the rate-limiting step for ATP hydrolysis under the conditions used. All these results support our previous conclusion [Shigekawa, M., Wakabayashi, S., & Nakamura, H. (1983) J. Biol. Chem. 258, 14157-14161] that the minimum requirement of divalent cations for the rapid turnover of the Ca2+-ATPase is 3 mol per mol of the enzyme active site, of which two are Ca2+ ions bound at the transport sites, and that the metal moiety of the metal-ATP complex bound at the ATP site determines the catalytic rate of the reaction steps.