Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ.

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to alpha-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate: other lectins had no such effects. Concanavalin A similar amounts partially inhibited (Na+ + K+)-ATPase; this inhibition was reversible by alpha-methylglucoside. There was no corresponding effect of concanavalin A on the potassium p-nitrophenylphosphatase. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na+ and/or ATP binding, but does not interact with a K+ site.

Sodium-potassium-activated adenosine triphosphatase of electrophorus electric organ. X. Immunochemical properties of the Lubrol-solubilized enzume and its constituent polypeptides.

Detergent (Lubrol WX)-solubilized sodium-potassium-activated adenosine triphosphatase ((Na+ + K+)-ATPase) of electrophorus electric organ contains two major constituent polypeptides with molecular weights of 96,000 and 58,000 which can be readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These two polypeptides can be clearly separated and can be obtained in milligram quantities by preparative sodium dodecyl sulfate gel electrophoresis. The separated polypeptides, after removal of sodium dodecyl sulfate, and Lubrol-solubilized (Na+ + K+)-ATPase activity to some degree. Moreover, the degree of inhibition is directly proportional to the increasing amounts of antisera. The inhibition is maximal 4 weeks after the first injection. Immunodiffusion in 1% agar gel indicated that only Lubrol-solubilized enzyme antiserum, but not 58,000-dalton or 96,00-dalton polypeptide antiserum, gives one major precipitin band. However, specific complex formation between each polypeptide antiserum and Lubrol-solubilized enzyme occurs. This was demonstrated indirectly. After incubating Lubrol-solubilized enzyme with increasing amounts of polypeptide antisera at 37 degrees for 15 min, they were placed in the side wells of an immunodiffusion plate with antiserum against Lubrol-solubilized enzyme in the central well. The intensity of the precipitin band decreased with increasing amounts of polypeptide antisera. Thus, the results indicate that both 96,000-dalton and 58,000-dalton polypeptides are integral subunits of (Na+ + K+)-ATPase.

Evidence for a new intermediate state in the mechanism of (Na+ + K+)-adenosine triphosphatase.

A rapid mixing technique was used to follow the intermediate formation of phosphorylated enzyme and liberation of inorganic phosphate by a microsomal preparation of (Na+ + K+)-ATPase. In the presence of 100 mM Na+,but without added K+, phosphorylation reaches a constant level at a rate which is dependent on ATP concentration. Inorganic phosphate production lags during the inital phase of phosphorylation and then accumulates at a constant rate. These observations favor a scheme in which Pi is liberated as the result of turnover of the phosphorylated enzyme. In the presence of 100 mM Na+ and 2.5 mM K+ phosphate production was resolved into two phases consisting of an initial 'burst' and late steady state phase...