Identification of the amino acids comprising a surface-exposed epitope within the nucleotide-binding domain of the na+, K+-ATPase using a random peptide library

Abstract

Monoclonal antibodies that bind native protein can generate considerable information about structure/function relationships, but identification of their epitopes can be problematic. Previously, monoclonal antibody M8‐P1‐A3 has been shown to bind to the catalytic (α) subunit of the Na⁺, K⁺‐ATPase holoenzyme and the synthetic peptide sequence 496‐HLLVMK^*GAPER‐506, which includes Lys 501 (K^*), the major site for fluorescein‐5′‐isothio‐cyanate labeling of the Na⁺, K⁺‐ATPase. This sequence region of α is proposed to comprise a portion of the enzyme's ATP binding domain (Taylor, W.R. & Green, N.W., 1989, Eur. J. Biochem. 179, 241–248). In this study we have determined M8‐P1‐A3′s ability to recognize the α‐subunit or homologous E₁E₂‐ATPase proteins from different species and tissues in order to deduce the antibody's epitope. In addition the bacteriophage random peptide or “epitope” library, recently developed by Scott and Smith (1990, Science 249, 386–390) and Devlin et al. (Devlin, J.J., Panganiban, L.C., & Devlin, P.E., 1990, Science 249, 404–406), has served as a convenient technique to confirm the species‐specificity mapping data and to determine the exact amino acid requirements for antibody binding. The M8‐P1‐A3 epitope was found to consist of the five amino acid 494–PRHLL‐498 sequence stretch of α, with residues PRxLx being critical for antibody recognition.