Ca2+, caldesmon, and myosin light chain kinase exchange with calmodulin.
Wheat calmodulin (CaM) was labeled at Cys-27 with the sulfhydryl-specific fluorescent probe 2-(4'-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS), to form MIANS.CaM. In the presence of Ca2+, MIANS.CaM undergoes a large fluorescence increase when it binds myosin light chain kinase (MLCK) and caldesmon (CaD), but little fluorescence change when it binds CaM antagonists or Ca2+. MLCK associates with MIANS.CaM at a rate of 2.8 x 10(7) M-1 s-1 and dissociates at 0.031 s-1 (Kd = 1.1 nM). Protein kinase A phosphorylation of MLCK (P-MLCK) produces a 3.5-fold decrease in its association rate with CaM and a 6-fold increase in its dissociation rate (Kd = 23 nM). CaD associates with MIANS.CaM with a rate of 5.3 x 10(8) M-1 s-1 and dissociates at 57 s-1 (Kd = 108 nM). EGTA disrupts the CaM.MLCK, CaM.P-MLCK, and the CaM.CaD complexes at rates of 3.5 s-1, 6.5 s-1, and 13.5 s-1, respectively. MLCK, therefore, dissociates from CaM more quickly by Ca2+ removal while the lower affinity CaD is dissociated more quickly by competition from higher affinity CaM target proteins than by Ca2+ removal. MLCK binding to CaM slowed Ca2+ dissociation from CaM's C-terminal Ca(2+)-binding sites from 30 s-1 to 6 s-1 while CaD had little effect on Ca2+ dissociation from these sites. During a Ca2+ transient, CaM could exchange with MLCK and CaD rapidly enough for these proteins to be directly involved in the contraction/relaxation cycle of smooth muscle.
Kasturi
R
R
Ohio State University Medical Center, Department of Medical Biochemistry, Columbus 43210.
Vasulka
C
C
Johnson
J D
JD
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D005456:Fluorescent Dyes; D005895:Gizzard, Avian; D007700:Kinetics; D009130:Muscle, Smooth / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D014908:Triticum / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D005456:Fluorescent Dyes; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D009219:Myosin-Light-Chain Kinase; D002118:Calcium