Anti-peptide monoclonal antibody imaging of a common binding domain involved in muscle regulation.
Multiple-component regulatory protein systems function through a generalized mechanism where a single regulatory protein or ligand binds to a variety of receptors to modulate specific functions in a physiologically sensitive context. Muscle contraction is regulated by the interaction of actin with troponin I (TnI) or myosin in a Ca(2+)-sensitive manner. Actin utilizes a single binding domain (residues 1-28) to bind to residues 104-115 of TnI (Van Eyk JE, Sönnichsen FD, Sykes BD, Hodges RS, 1991, In: Rüegg JC, ed, Peptides as probes in muscle research, Heidelberg, Germany: Springer-Verlag, pp 15-31) and to myosin subfragment 1 (S1, an enzymatic fragment of myosin containing both the actin and ATP binding sites) (Van Eyk JE, Hodges RS, 1991, Biochemistry 30:11676-11682) in a Ca(2+)-sensitive manner. We have utilized an anti-TnI peptide (104-115) monoclonal antibody, Mab B4, that binds specifically to TnI, to image the common binding domain of actin and thus mimic the activity of actin including activation of the S1 ATPase activity and TnI-mediated regulation of the S1 ATPase. Mab B4 has also been utilized to identify a receptor binding domain on myosin (residues 633-644) that is recognized by actin. Interestingly, Mab B4 binds to the native protein receptors TnI and S1 with relative affinities of 100- and 25,000-fold higher than the binding affinity to the 12-residue peptide immunogen. Thus, anti-peptide monoclonal antibodies prepared against a receptor binding domain can mimic the ligand binding domain and be utilized as a powerful tool for the detailed analysis of complex multiple-component regulatory systems.
Van Eyk
J E
JE
Department of Biochemistry and Medical Research Council of Canada Group in Protein Structure and Function, University of Alberta, Edmonton, Canada.
Caday-Malcolm
R A
RA
Yu
L
L
Irvin
R T
RT
Hodges
R S
RS
D000199:Actins / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002642:Chick Embryo; D002645:Chickens; D017124:Conserved Sequence; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000737:chemistry; D009218:Myosins / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits; D014336:Troponin / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D019210:Troponin I
D000199:Actins; D000911:Antibodies, Monoclonal; D010455:Peptides; D014336:Troponin; D019210:Troponin I; D009218:Myosins; D002118:Calcium