Occurrence and role of tightly bound adenine nucleotides in sarcoplasmic reticulum of rabbit skeletal muscle.

Abstract

Freshly isolated sarcoplasmic reticulum vesicles contain 0.05 mol of tightly bound ADP and 0.03 mol of tightly bound ATP/mol of Ca2+,Mg2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). These values were increased to 0.1-0.2 mol ADP and 0.2-0.3 mol ATP/mol of ATPase after incubation of vesicles in the presence of MgATP and Ca2+ at 25.degree. C and pH 7.0. Half-maximal enrichment of tightly bound nucleotides was obtained with 2.5 mM ATP and 0.32 .mu.M free Ca2+. Uncoupling of Ca transport from ATPase activity by mild acidic conditions or with ethylene glycol bis(.beta.-aminoethyl ether)N,N,N'',N''-tetraacetic acid at pH 7.0 decreased the ability of the membranes to be enriched with tightly bound nucleotides and also decreased the content of tightly bound nucleotides of previously enriched membranes. Tightly bound [3H]nucleotides could only be partially displaced by reincubation under enrichment conditions. Tightly bound nucleotides are associated with energized Ca translocation but do not appear to be directly involved in the catalytic cycle.