Identification of monoclonal antibody 4A-binding site on the transducin alpha subunit. Immunoblotting of submaxillary Arg-C protease fragments of transducin.
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.
Hingorani
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Department of Biological Chemistry, University of Illinois, Chicago 60612.
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D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology*; D010449:Peptide Mapping; D012374:Rod Cell Outer Segment / Q000737:chemistry; D012697:Serine Endopeptidases; D014108:Tosylphenylalanyl Chloromethyl Ketone; D015293:Transducin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D000246:Adenosine Diphosphate Ribose; D014108:Tosylphenylalanyl Chloromethyl Ketone; D010450:Endopeptidases; C044499:submandibular proteinase A; D012697:Serine Endopeptidases; D015293:Transducin