title: [Effect of preincubation on smooth muscle myosin light chain kinase activity].
abstract: Phosphorylation of myosin regulatory light chain (RLC) catalysed by myosin light chain kinase (MLCK) is a key reaction in the regulation of actin-myosin interaction in smooth muscle. The activation of MLCK by calmodulin (CaM) and Ca2+ was investigated over a wide range of the enzyme concentrations using myosin or its RLC with Mw = 20 kDa as substrates. Kinase activation by CaM (at saturating Ca2+ concentrations) was characterized by positive cooperativity even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between MLCK and CaM. The activation of the kinase by Ca2+ was also cooperative but only at relatively low CaM levels. This cooperativity was shown to result from time dependent changes in MLCK that take place during its incubation with Ca2+ and CaM before substrate addition in phosphorylation assays. As a result the kinase activity as a function of its concentration at constant CaM level was biphasic: there was the activity optimum at 1:1 ratio of CaM to MLCK and almost complete inhibition at 3 to 7 molar excess of kinase over CaM. Such changes that take place during 10 to 15 min preincubation with Ca2+ and CaM may involve the kinase supramolecular structure formation or/and its conformational rearrangements.
authors.affiliation: Taras Shevchenko Kiev University, Institute of Physiology.
authors.forename: A M
authors.initials: AM
authors.lastname: Filenko
authors.forename: A
authors.initials: A
authors.lastname: Sobieszek
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D004789:Enzyme Activation; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D014421:Turkey
chemical_list: D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase
vernacular_title: Zalezhnist' aktyvnosti kinazy lehkykh lantsiuhiv miozynu hladen'kykh m'iaziv vid umov peredinkubatsiï.