Uncoupling the red cell sodium pump by proteolysis.

摘要: Abstract In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This evident both absence intravesicular alkali cations when Na-ATPase compared to ATP-dependent 22Na+ influx, and presence K+ Na+/K+ exchange (Na+ + K+)-activated ATPase. differential loss pump versus hydrolysis observed also activities only intact, non-leaky vesicles were therefore reflects intramolecular uncoupling rather nonspecific leakage. Although oligomycin thimerosal, like chymotrypsin, inhibit enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) Na,K-ATPase, at least as it exists situ, alters reaction sequence respect normal ordered mechanism. Accordingly, cytoplasmic Na+ extracellular bind enzyme, stimulate phosphorylation (ATP E1----E1P ADP) dephosphorylation (E2P----E2 Pi), respectively, but each then released same side from which had bound; presumably release occurs prior transitions E1P E2P E2 E1. conclusion supported by experiments showing that, ar micromolar concentration, hydrolytic activity (Na-ATPase) trypsinized not unmodified stimulated K+, consistent earlier (Hegyvary, C., Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) K X E1 transition slower transition.