The Steady State Level of Phosphorylated Intermediate in Relation to the Two Sodium-dependent Adenosine Triphosphatases of Calf Brain Microsomes

During ATP hydrolysis, microsomal preparations form an acid-stable protein-phosphate complex. Since there is evidence that two different independent enzymatic sites catalyze the hydrolysis of ATP in calf brain microsomes, it was reasoned that either one or both of these might contribute to the formation of this complex. To determine the actual relationships, the phosphorylation reaction was compared with hydrolysis at each of the ATPase sites—at different levels of Na+, K+, and ATP and at different temperatures. The kinetic analysis indicates that it is hydrolysis at Site I, the Na+, -K+ ATPase, which correlates with the steady state level of protein-phosphate complex. The results add support to the hypothesis that this complex is an intermediate in the hydrolysis of ATP by the Na+-K+ ATPase—the site that takes part in Na+ and K+ transport across biological membranes. The second ATPase, Site II, is a Na+-ATPase inhibited by K+; the kinetic analysis indicates that this site does not contribute substantially to the steady state level of acid-stable phosphorylated intermediate. It is suggested that Site II may be related to a sodium pump such as the one in red blood cells described by J. F. Hoffman and F. M. Kregnow (Ann. N. Y. Acad. Sci., 137, 566 (1966)).