IDENTIFICATION OF THE AMINO-ACIDS COMPRISING A SURFACE-EXPOSED EPITOPE WITHIN THE NUCLEOTIDE-BINDING DOMAIN OF THE NA-ATPASE USING A RANDOMPEPTIDE LIBRARY(,K+)

Monoclonal antibodies that bind native protein can generate considerab le information about structure/function relationships, but identificat ion of their epitopes can be problematic. Previously, monoclonal antib ody M8-P1-A3 has been shown to bind to the catalytic (alpha) subunit o f the Na+,K+-ATPase holoenzyme and the synthetic peptide sequence 496- HLLVMKGAPER-506, which includes Lys 501 (K*), the major site for fluo rescein-5'-isothiocyanate labeling of the Na+,K+-ATPase. This sequence region of alpha is proposed to comprise a portion of the enzyme's ATP binding domain (Taylor, W.R. & Green, N.W., 1989, Eur. J. Biochem. 17 9, 241-248). In this study we have determined M8-P1-A3's ability to re cognize the alpha-subunit or homologous E1E2-ATPase proteins from diff erent species and tissues in order to deduce the antibody's epitope. I n addition the bacteriophage random peptide or ''epitope'' library, re cently developed by Scott and Smith (1990, Science 249, 386-390) and D evlin et al. (Devlin, J.J., Panganiban, L.C., & Devlin, P.E., 1990, Sc ience 249, 404-406), has served as a convenient technique to confirm t he species-specificity mapping data and to determine the exact amino a cid requirements for antibody binding. The M8-P1-A3 epitope was found to consist of the five amino acid 494-PRHLL-498 sequence stretch of al pha, with residues PRxLx being critical for antibody recognition.