ANTIPEPTIDE MONOCLONAL-ANTIBODY IMAGING OF A COMMON BINDING DOMAIN INVOLVED IN MUSCLE REGULATION

Multiple-component regulatory protein systems function through a gener alized mechanism where a single regulatory protein or ligand binds to a variety of receptors to modulate specific functions in a physiologic ally sensitive context. Muscle contraction is regulated by the interac tion of actin with troponin I (TnI) or myosin in a Ca2+-sensitive mann er. Actin utilizes a single binding domain (residues 1-28) to bind to residues 104-115 of TnI (Van Eyk JE, Sonnichsen FD, Sykes ED, Hedges R S, 1991, In: Ruegg JC, ed, Peptides as probes in muscle research, Heid elberg, Germany: Springer-Verlag, pp 15-31) and to myosin subfragment I (SI, an enzymatic fragment of myosin containing both the actin and A TP binding sites) (Van Eyk JE, Hedges RS, 1991, Biochemistry 30:11676- 11682) in a Ca2+-sensitive manner. We have utilized an anti-Tnf peptid e (104-115) monoclonal antibody, Mab B4, that binds specifically to Tn I, to image the common binding domain of actin and thus mimic the acti vity of actin including activation of the S1 ATPase activity and TnI-m ediated regulation of the S1 ATPase. Mab B4 has also been utilized to identify a receptor binding domain on myosin (residues 633-644) that i s recognized by actin. Interestingly, Mab B4 binds to the native prote in receptors TnI and S1 with relative affinities of 100- and 25,000-fo ld higher than the binding affinity to the 12-residue peptide immunoge n. Thus, anti-peptide monoclonal antibodies prepared against a recepto r binding domain can mimic the ligand binding domain and be utilized a s a powerful tool for the detailed analysis of complex multiple-compon ent regulatory systems.