Identification of monoclonal antibody 4A-binding site on the transducin alpha subunit. Immunoblotting of submaxillary Arg-C protease fragments of transducin

• Published in: The Journal of biological chemistry 1990-11, Vol.265 (32), p.19923-19927
• Main Authors: Hingorani, V N, Ho, Y K
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate Ribose - metabolism; Analytical, structural and metabolic biochemistry; Animals; Antibodies, Monoclonal - metabolism; Binding and carrier proteins; Binding Sites, Antibody; Biological and medical sciences; Blotting, Western; Cattle; Endopeptidases - metabolism; Epitopes - immunology; Fundamental and applied biological sciences. Psychology; Molecular Weight; Peptide Fragments - immunology; Peptide Mapping; Proteins; Rod Cell Outer Segment - chemistry; Serine Endopeptidases; Tosylphenylalanyl Chloromethyl Ketone; Transducin - immunology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)45461-5

Monoclonal antibody 4A (mAb 4A) against the T alpha subunit of transducin has been widely used to study the structure and function of signal transducing GTP-binding proteins involved in the regulation of visual excitation, hormonal regulation of adenylyl cyclase and ionic channels. Results of mapping the epitope-binding site of mAb 4A on T alpha have been controversial. Hamm and co-workers (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) reported that mAb 4A interacts with T alpha at the carboxyl-terminal peptide, whereas Fung and co-workers (Navon, S. E., and Fung, B. K.-k. (1988) J. Biol. Chem. 263, 489-498) showed that mAb 4A binds mainly at the amino-terminal peptide. In this report, we examine the epitope-binding site of mAb 4A by Western immunoblotting of the proteolytic fragments of T alpha generated by submaxillary Arg-C protease digestion. Submaxillary Arg-C protease cleaved T alpha at two sites, Arg-204 and Arg-310, generating two major fragments of apparent size 35 (T alpha'SM-35) and 23 kDa (T alpha'SM-23). Both fragments contain the amino-terminal peptide of T alpha but lack the carboxyl-terminal peptide. Western immunoblotting showed that mAb 4A cross-reacted with both peptides. Treatment of T alpha'SM-35 and T alpha'SM-25 with L-1-(tosylamido)-2-phenyethyl chloromethyl ketone-trypsin removed the amino-terminal 2-kDa peptide with concomitant loss of mAb 4A reactivity. This observation unequivocally confirms the result of Fung and co-workers that the epitope for mAb 4A is located on the amino-terminal 2-kDa peptide of T alpha. This conclusion should provide a more accurate interpretation of results in the literature as well as of future studies in which mAb 4A is used.