Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase.
A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D000939:Epitopes; D009219:Myosin-Light-Chain Kinase