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

被引:13
作者
MALIK, B
JAMIESON, GA
BALL, WJ
机构
[1] UNIV CINCINNATI, COLL MED, DEPT PHARMACOL & CELL BIOPHYS, CINCINNATI, OH 45267 USA
[2] UNIV CINCINNATI, COLL MED, DEPT ENVIRONM HLTH, CINCINNATI, OH 45267 USA
关键词
BACTERIOPHAGE RANDOM PEPTIDE LIBRARY; EPITOPE; MONOCLONAL ANTIBODY; NA+; K+-ATPASE;
D O I
10.1002/pro.5560021211
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:2103 / 2111
页数:9
相关论文
共 40 条
[1]   THE INHIBITORY MONOCLONAL-ANTIBODY M7-PB-E9 STABILIZES E(2) CONFORMATIONAL STATES OF NA+,K+-ATPASE [J].
ABBOTT, A ;
BALL, WJ .
BIOCHEMISTRY, 1992, 31 (45) :11236-11243
[2]   THE EPITOPE FOR THE INHIBITORY ANTIBODY M7-PB-E9 CONTAINS SER-646 AND ASP-652 OF THE SHEEP NA+,K+-ATPASE ALPHA-SUBUNIT [J].
ABBOTT, A ;
BALL, WJ .
BIOCHEMISTRY, 1993, 32 (13) :3511-3518
[3]   IMMUNOCHEMICAL AND SPECTROSCOPIC CHARACTERIZATION OF 2 FLUORESCEIN 5'-ISOTHIOCYANATE LABELING SITES ON NA+,K+-ATPASE [J].
ABBOTT, AJ ;
AMLER, E ;
BALL, WJ .
BIOCHEMISTRY, 1991, 30 (06) :1692-1701
[4]  
AUSUBEL FA, 1989, VECTORS DERIVED PLAS, V1
[5]   IMMUNOCHEMICAL EVIDENCE THAT THE FITC-LABELING SITE ON NA+,K+-ATPASE IS NOT THE ATP BINDING-SITE [J].
BALL, WJ ;
FRIEDMAN, ML .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1987, 148 (01) :246-253
[6]   ISOLATION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES TO (NA+ + K+)-ATPASE [J].
BALL, WJ ;
SCHWARTZ, A ;
LESSARD, JL .
BIOCHIMICA ET BIOPHYSICA ACTA, 1982, 719 (03) :413-423
[7]   IMMUNOCHEMICAL COMPARISON OF CARDIAC GLYCOSIDE-SENSITIVE (LAMB) AND GLYCOSIDE-INSENSITIVE (RAT) KIDNEY (NA++K+)-ATPASE [J].
BALL, WJ ;
LANE, LK .
BIOCHIMICA ET BIOPHYSICA ACTA, 1986, 873 (01) :79-87
[9]   IMMUNOCHEMICAL STUDIES OF (NA++K+)-ATPASE USING SITE-SPECIFIC, SYNTHETIC PEPTIDE DIRECTED ANTIBODIES [J].
BALL, WJ ;
LOFTICE, CD .
BIOCHIMICA ET BIOPHYSICA ACTA, 1987, 916 (01) :100-111
[10]   STUDIES OF THE ANTIGENIC PROPERTIES OF SHEEP KIDNEY NA+,K+-ATPASE [J].
BALL, WJ ;
SCHWARTZ, A .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1982, 217 (01) :110-119