Activation of smooth muscle myosin light chain kinase activity by a monoclonal antibody which recognizes the calmodulin-binding region.
The regulatory domain of smooth muscle myosin light chain kinase (MLCK) was studied using monoclonal antibodies. Of the 22 monoclonal antibodies tested, a monoclonal antibody designated LKH-18 was found to activate MLCK in the absence of Ca2+/calmodulin. This activation was even greater when an Fab fragment of LKH-18 was used. Consequently, the actin-dependent smooth muscle myosin ATPase activity and the superprecipitation of actomyosin were significantly activated by MLCK plus LKH-18, even in the absence of Ca2+/calmodulin. The antibody-binding site was studied using proteolytic fragments and synthetic peptide analogues of MLCK. Immunoblot analysis revealed that LKH-18 reacted with the 66 kDa calmodulin-dependent active fragment but not with the 64 kDa inactive fragment or with the 61 kDa calmodulin-independent active fragment. Furthermore, LKH-18 reacted with MLCK-(796-815)-peptide but not with MLCK-(786-801)-peptide or with MLCK-(796-807)-peptide. Therefore the LKH-18-binding site was assigned to amino acid residues 808-815 of MLCK, which are thought to be a part of the calmodulin-binding site. The present results suggest that the binding of ligand to this region induces a conformation change in MLCK and that this abolishes the action of the inhibitory region which exists next to the N-terminal side of the calmodulin-binding site.
Araki
Y
Y
Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, OH 44106.
Ikebe
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D004789:Enzyme Activation; D000939:Epitopes / Q000276:immunology; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D014422:Turkeys
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D002118:Calcium