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

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Abstract
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 α (catalytic) and the β (glycoprotein) subunits of Na/K-ATPase. The titers of the anti-α and anti-β 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-α subunit from anti-β subunit antibodies. There was not cross-reactivity of antibodies to one subunit with the other subunit. The α 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-α antibodies from all of the rabbits reacted with both proteolytic fragments. The anti-guinea pig Na/K-ATPase antisera (pooled) cross-reacted with the α subunit of Na/K-ATPase from human, cow, dog, rabbit, rat mouse, turtle, and toad; and with the β 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 antisera 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 α chain was 96,000 daltons. Its identity was established with purified anti-α antibodies and by immunocompetition with purified Na/K-ATPase and Ca-ATPase. Translation of the β subunit was not detected in this system.