Monoclonal antibody assessment of tissue- and species-specific myosin light chain kinase isozymes.
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.
Hagiwara
M
M
Department of Pharmacology, Nagoya University School of Medicine, Aichi.
Tokumitsu
H
H
Onoda
K
K
Tanaka
T
T
Ito
M
M
Kato
N
N
Hidaka
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D000939:Epitopes / Q000032:analysis; D005895:Gizzard, Avian / Q000201:enzymology; D015151:Immunoblotting; D007527:Isoenzymes / Q000032:analysis / Q000276:immunology*; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000032:analysis / Q000276:immunology*; D013045:Species Specificity; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007527:Isoenzymes; D009219:Myosin-Light-Chain Kinase