Characteristics of antibodies to guinea pig (Na+ + K+)-adenosine triphosphatase and their use in cell-free synthesis studies.

Antibodies have been produced, in three rabbits, to Na/K-ATPase purified from guinea pig renal outer medulla. Each rabbit produced antibodies to both the alpha (catalytic) and the beta (glycoprotein) subunits of Na/K-ATPase. The titers of the anti-alpha and anti-beta antibodies varied with time and between rabbits. None of the antisera inhibited Na/K-ATPase activity under various preincubation conditions. A method is presented for separating small amounts of anti-alpha subunit from anti-beta subunit antibodies. There was no cross-reactivity of antibodies to one subunit with the other subunit. The alpha subunit of the Na/K-ATPase was cleaved into a 41,000-dalton peptide (that contains the ATP phosphorylating site) and a 58,000-dalton hydrophobic peptide as described by Castro and Farley (Castro, J., Farley, R.A., 1979, J. Biol. Chem. 254: 2221-2228). Anti-alpha antibodies from all of the rabbits reacted with both proteolytic fragments. The anti-guinea pig Na/K-ATPase antisera (pooled) cross-reacted with the alpha subunit of Na/K-ATPase from human, cow, dog, rabbit, rat, mouse, turtle, and toad; and with the beta subunit from human, rat, and mouse. The loci of cross-reactivity were investigated using partially purified canine kidney Na/K-ATPase cleaved with trypsin as described above. The anti-sera from rabbits 1 and 2 cross-reacted with the 41,000-dalton peptide from the dog but very little with the 58,000-dalton peptide. No cross-reactivity was observed with antiserum from rabbit 3 to either fragment. Guinea pig kidney RNA was translated in a rabbit reticulocyte lysate system followed by immunoprecipitation with the antisera. The molecular weight of the cell-free synthesized alpha chain was 96,000 daltons. Its identity was established with purified anti-alpha antibodies and by immunocompetition with purified Na/K-ATPase and Ca-ATPase. Translation of the beta subunit was not detected in this system.