Hagiwara M, Tokumitsu H, Onoda K, Tanaka T, Ito M, Kato N, Hidaka H
Department of Pharmacology, Nagoya University School of Medicine, Aichi.
J Biochem. 1989 Jul;106(1):71-5. doi: 10.1093/oxfordjournals.jbchem.a122822.
Monoclonal antibodies raised against chicken gizzard smooth muscle myosin light chain kinase were used for immunological and structural studies of this enzyme. Epitope mapping of trypsin-digested chicken gizzard enzyme showed that MM-1, 2, 3, 4, 5, 6, and 7 bind to 65 kDa (trypsin-digested) and 60 kDa (chymotrypsin-digested) fragments which contain the catalytic domain of the kinase. Kinetic analysis demonstrated that MM-7 inhibited kinase activity competitively with respect to ATP and noncompetitively with respect to myosin light chain, thereby indicating that MM-7 binds at or near the ATP binding site of the enzyme. Immunoblot analysis revealed that all these antibodies (MM-1 to 12) reacted with the enzyme (130 kDa) from intestinal and vascular smooth muscles, whereas 5 (MM-1, 3, 4, 6, and 9) or 3 (MM-1, 3, and 4) of 12 antibodies did not cross-react with chicken cardiac muscle or with blood platelet myosin light chain kinase (130 kDa), respectively. None of these antibodies showed cross-reactivity against skeletal muscle myosin light chain kinase. As for mammalian species, MM-11 and 12 reacted with myosin light chain kinase of vascular smooth muscle (140 kDa) and MM-11 cross-reacted with the enzyme (140 kDa) from cardiac muscle of rat and rabbit. These data suggest the existence of at least 4 subspecies of myosin light chain kinase in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.
