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J. Biochem, 1989, Vol. 106, No. 1 71-75
© 1989 Japanese Biochemical Society

research-article

Monoclonal Antibody Assessment of Tissue- and Species-Specific Myosin Light Chain Kinase Isozymes1

Masatoshi Hagiwara*, Hiroshi Tokumitsu*, Koji Onoda*,2, Toshio Tanaka*,3, Masaaki Ito*,4, Nobuo Kato** and Hiroyoshi Hidaka*,5

*Department of Pharmacology Showa-ku, Nagoyat Aichi 466
**Department of Bacteriology, Nagoya University School of Medicine Showa-ku, Nagoyat Aichi 466

5Departments of Thoracic Surgery, Edobashi, Tsu, Mie 514.

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.

1This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

2Departments of Thoracic Surgery, Mie University School of Medicine, Edobashi, Tsu, Mie 514

3Molecular and Cellular Pharmacology, Mie University School of Medicine, Edobashi, Tsu, Mie 514

4Internal Medicine, Mie University School of Medicine, Edobashi, Tsu, Mie 514


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