The epitope for the inhibitory antibody M7-PB-E9 contains Ser-646 and Asp-652 of the sheep Na+,K(+)-ATPase alpha-subunit

Abstract

The binding of monoclonal antibody M7-PB-E9 to the alpha-subunit of Na+,K(+)-ATPase partially inhibits enzyme activity (35%) in competition with ATP, while in the presence of magnesium it stimulates the rate of ouabain binding severalfold [Ball, W. J. (1984) Biochemistry 23, 2275-2281]. These effects have been shown to result from an antibody-induced shifting of the enzyme's E1 <==> E2 conformational equilibrium to the right that affects all enzyme-ligand interactions except that with Mg2+ [Abbott, A.J., & Ball, W.J. (1992) Biochemistry 31, 11236-11243]. In order to identify the location of the M7-PB-E9 epitope, proteolytic fragments of the lamb kidney enzyme were generated and the immunoreactive alpha fragments were identified by Western blot analyses. These studies revealed a 47-kDa tryptic fragment, which bound both M7-PB-E9 and a -COOH terminus specific antisera and NH2-terminal sequencing showed to originate at Ala-590. Digestion with Staphylococcus aureus V8 protease produced a 36-kDa -COOH-terminus fragment which originated at Gly-697 and did not contain the antibody epitope. Thus the intracellular sequence region Ala-590 to Gly-697 was shown to contain the antibody epitope. When M7-PB-E9's ability to recognize the alpha subunits from various species and tissues was determined and correlated with available sequencing data, only Ser-646 was present in the highly reactive lamb, pig, and avian kidney alpha 1 proteins and altered (Asn) in the poorly recognized Xenopus and rat kidney and Torpedo electroplax organ enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)