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.
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 (alpha) 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'-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. 179, 241-248). In this study we have determined M8-P1-A3's ability to recognize the alpha-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 alpha, with residues PRxLx being critical for antibody recognition.
Malik
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B
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio 45267-0575.
Jamieson
G A
GA
Ball
W J
WJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D000939:Epitopes / Q000276:immunology*; D017912:Inovirus / Q000235:genetics; D007679:Kidney Medulla / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D013045:Species Specificity; D013499:Surface Properties; D013552:Swine; D014101:Torpedo; D014981:Xenopus
D000939:Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase