Smooth muscle myosin as a calmodulin binding protein. Affinity increase on filament assembly.
Smooth muscle myosin is normally copurified with myosin light chain kinase (MLCKase) and calmodulin (CM). We have now established the binding affinities and stoichiometries of these two components with respect to monomeric and filamentous myosin. The relative amounts of CM and MLCKase in fresh synthetic myosin filaments were approximately stoichiometrical but for both in a molar ratio to myosin of about 1 to 30 or less. A 10(7) dilution of filaments did not result in any significant decrease in the amount of endogenous MLCKase and CM except in the absence of Ca2+ when the CM content was reduced around five-fold. Binding assays were performed with myosin depleted of CM and MLCKase by passage over melittin- and CM-affinity columns, arranged in tandem. For binding to myosin preassembled into filaments three classes of CM binding sites could be demonstrated. (1) A high affinity binding characterized by a dissociation constant of 20-30 nM and a rather low binding stoichiometry of below 1 to 500. (2) An intermediate affinity, characterized by a dissociation constant of 1.2 microM and 1 to 100 binding stoichiometry. (3) A low affinity with a Kd greater than 10 microM and with an approximate 1 to 1 binding ratio relative to myosin. If CM was made available during filament assembly the high affinity binding predominated, with a stoichiometry in the presence of Ca2+ of about 1 to 50. The binding affinity but not the stoichiometry was reduced several fold by the removal of Ca, excluding a non-specific trapping of CM within the filament architecture. Collectively, these data demonstrate an independent and specific association of MLCKase and CM with myosin, that is strengthened by filament assembly.
Sobieszek
A
A
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
D000818:Animals; D001704:Biopolymers; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian; D007700:Kinetics; D009130:Muscle, Smooth / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000032:analysis; D009218:Myosins / Q000378:metabolism*; D013270:Stomach; D013552:Swine; D014422:Turkeys
D001704:Biopolymers; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D009219:Myosin-Light-Chain Kinase; D009218:Myosins