Anti-gizzard MLCK monoclonal antibody MM13 inhibits superprecipitation and phosphorylation of bovine aortic smooth muscle actomyosin.
We prepared monoclonal antibodies directed against chicken gizzard myosin light chain kinase (MLCK) and used them to study the contractile system of aortic smooth muscle. One monoclonal antibody, MM13, dose dependently inhibited actomyosin superprecipitation of bovine aortic smooth muscle, in accord with the suppression of 20 kDa myosin light chain phosphorylation by endogenous kinase. Immunoblotting analysis demonstrated that MM13 cross-reacted with the 150,000 Mr peptide of bovine aortic actomyosin preparation. The bovine aortic MLCK was purified approximately 2,400-fold to apparent homogeneity by three steps of column chromatography. The purified enzyme has a molecular weight of 150,000 and a slower mobility than chicken gizzard MLCK (130,000 Mr), as determined by SDS-polyacrylamide gel electrophoresis. MM13 also cross-reacted with purified bovine aortic MLCK and inhibited the kinase activity, in vitro. We interpret these findings to mean that binding of the anti-gizzard MLCK monoclonal antibody directly to aortic smooth muscle MLCK (150,000 Mr) decreases the phosphorylation of the 20 kDa myosin light chain, thus suppressing the aortic smooth muscle myosin-actin interaction.
Tokumitsu
H
H
Department of Pharmacology, Nagoya University School of Medicine, Aichi.
Hagiwara
M
M
Onoda
K
K
Hidaka
H
H
D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001011:Aorta / Q000378:metabolism*; D002417:Cattle; D011232:Chemical Precipitation; D002645:Chickens; D003429:Cross Reactions; D005895:Gizzard, Avian / Q000201:enzymology*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation
D000911:Antibodies, Monoclonal; D000205:Actomyosin; D009219:Myosin-Light-Chain Kinase