A Phosphorylated Intermediate in Adenosine Triphosphate-dependent Sodium and Potassium Transport across Kidney Membranes

Abstract

(Received for publication, September 21, 1964) An enzymatic system in cell membranes actively transports sodium ions outward and potassium ions inward. Like other enzyme systems this one has a st,oichiometry and presumably, a reaction sequence and active sites. Knowledge of the latter feat,ures is fragmentary. Isolation of a reaction intermediate would clarify the reaction sequence and could lead to chemical characterization of an active site. Active sodium and potassium transport in the human erythro- cyte is a stoichiometric reaction in which three sodium ions move outward, two potassium ions move inward, and one terminal phosphate bond of adenosine triphosphate is hydrolyzed at the inner surface of the membrane in the presence of magnesium ion (1). It is inhibited by cardiac glycosides such as ouabain. It appears in broken membranes as an ATPase activity which is dependent upon sodium plus potassium, and is sensitive to ouabain (2, 3). This will be abbreviated as (Na+ + K+)- ATPase in this paper. There is high (Na+ + K+)-ATPase ac- tivity in membrane preparations of many excitable and secret- ing tissues, particularly, electric organ (4), brain (5), peripheral nerve (6)) salt gland (7), kidney (5), and salivary gland (8). In 1957, Glynn (9) presented a scheme of Shaw to account for the stoichiometry of sodium and potassium transport by an alter- nation of carrier forms. Metabolic energy, now known to come from ATP, would convert a potassium carrier, X, into a sodium carrier, Y, on the inner face of the membrane. After outward translocation in combination with sodium ion, Y would revert to X on the outer face and, in combination with potassium ion, return through the membrane to the initial state. The scheme is consistent with reciprocal competitive inhibitory effects be- tween sodium ion and potassium ion (10). On the basis of tracer exchange experiments with (Na+ + K+)-ATPase in crab nerve, Skou (11) proposed an enzyme-high energy phosphate inter- mediate, E - P. His scheme was