THE EPITOPE FOR THE INHIBITORY ANTIBODY M7-PB-E9 CONTAINS SER-646 AND ASP-652 OF THE SHEEP NA+,K+-ATPASE ALPHA-SUBUNIT

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 a 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 alpha1 proteins and altered (Asn) in the poorly recognized Xenopus and rat kidney and Torpedo electroplax organ enzymes. In addition, M7-PB-E9 was found to have a high binding affinity for the rat alpha3 isoform and low affinity for alpha2 which is altered at Asp-652 --> Glu. The M7-PB-E9 epitope therefore was found to encompass the 7 amino acid sequence from Ser-636 to Asp-652. This site plays an important role in the E1 <-> E2 conformational transitions undergone by the enzyme and it lies within a unique domain that links the highly conserved nucleotide binding and hinge domains of alpha as proposed by MacLennan et al. [(1985) Nature 316, 696] for the homologous sarcoplasmic reticulum Ca2+, Mg2+-ATPase.