Selective binding of L-thyroxine by myosin light chain kinase.
L-Thyroxine selectively inhibited Ca2+-calmodulin-activated myosin light chain kinases (MLC kinase) purified from rabbit skeletal muscle, chicken gizzard smooth muscle, bovine thyroid gland, and human platelet with similar Ki values (Ki = 2.5 microM). A detailed analysis of L-thyroxine inhibition of smooth muscle myosin light chain kinase activation was undertaken in order to determine the effect of L-thyroxine on the stoichiometries of Ca2+, calmodulin, and the enzyme in the activation process. The kinetic data indicated that L-thyroxine does not interact with calmodulin but, instead, through direct association with the enzyme, inhibits the binding of the Ca2+-calmodulin complex to MLC kinase. L-[125I]Thyroxine gel overlay revealed that the 95-kDa fragment of chicken gizzard MLC kinase digested by chymotrypsin and all the fragments of 110, 94, 70, and 43 kDa produced by Staphylococcus aureus V8 protease digestion which contain the calmodulin binding domain retain L-[125I]thyroxine binding activity, whereas smaller peptides were not radioactive. Since MLC kinase is phosphorylated by cAMP-dependent protein kinase (2 mol of phosphate/mol of MLC kinase), the effect of L-thyroxine on the phosphorylation of MLC kinase also was examined. L-Thyroxine binding did not inhibit the phosphorylation of MLC kinase and, moreover, reversed the inhibition of phosphorylation obtained with the calmodulin-enzyme complex. These observations support the suggestion that L-thyroxine binds at or near the calmodulin-binding site of MLC kinase. L-Thyroxine may serve as a different type of pharmacological tool for elucidating the biological significance of MLC kinase-mediated reactions.
Hagiwara
M
M
Department of Pharmacology, Nagoya University School of Medicine, Japan.
Mamiya
S
S
Hidaka
H
H
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D002417:Cattle; D002645:Chickens; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009130:Muscle, Smooth / Q000201:enzymology; D009132:Muscles / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D011817:Rabbits; D013961:Thyroid Gland / Q000201:enzymology; D013974:Thyroxine / Q000378:metabolism / Q000494:pharmacology*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D009219:Myosin-Light-Chain Kinase; D013974:Thyroxine