Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

D O Souza,L De Meis

doi:10.1016/s0021-9258(20)81868-7

Abstract

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.

Highlights

In intact vesicles, which are able to accumulate Ca*+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca*’ concentration is increased to 10 mM
Evidence was presented that the nucleoside triphosphatase activity is inhibited by the saturation of a Ca2+-binding site of low affinity (K, 1 to 3 mM), located on the inner surface of the membrane [3,4,5] and that the different nucleoside triphosphates, besides phosphorylating the enzyme, can activate the rate of E m P hydrolysis [6, 7]
Leaky Vesicles-In the first experiment (Fig. 1) intact vesicles were incubated with ATP or ITP in media containing different

Summary

Introduction

In intact vesicles, which are able to accumulate Ca*+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca*’ concentration is increased to 10 mM. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added MgZ+ When these vesicles are incubated in media containing ITP and 0.1 mM Ca’+, addition of Mg*+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When the Ca *+ concentration is raised to 10 or 20 mM,‘the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg *+ In these conditions the ATPase and ITPase activities are activated by Mg’+, not to the level observed in 0.1 mM Ca’+. The role of Mg’+ in the nucleoside triphosphatase activity and steady state level of phosphoenzyme was investigated

Methods

Results

Conclusion