Abstract
The interaction between bovine testes calmodulin and rabbit fast skeletal muscle myosin light chain kinase was investigated with the zero-length cross-linking reagent N,N'-dicyclohexylcarbodiimide. A cross-linked product of 110 kDa was produced only in the presence of Ca2+. The reaction mixture was separated on diethylaminoethyl cellulose, and a fraction containing the cross-linked complex of calmodulin and myosin light chain kinase was found to have an elevated kinase activity in the absence of Ca2+, which constituted approximately 50% of the maximally stimulated kinase activity of control, and additional kinase activity in the presence of Ca2+, which constituted the remaining 50% of control activity. Calmodulin added exogenously to the cross-linked complex had no effect on the measured Ca2+ dependence or the maximal extent of kinase activity, which is consistent with the cross-linking of calmodulin in close proximity to a regulatory region of myosin light chain kinase. Moreover, the results are consistent with a mechanism whereby the association of calmodulin is sufficient to stimulate kinase activity and the binding of Ca2+ to bound calmodulin increases catalytic efficiency.
Highlights
The interaction between bovine testes calmodulin 1987; Kennelly et al, 1987; Pearson et al, 1988)
Calmodulin addedexogenously complexes with enzymes (Kincaid, 1984; Yamamoto et al, to the cross-linked complex had no effect on the meas- 1988).In thispaper, the Ca2+-dependentcovalent association ured Ca2+dependence or the maximal extent of kinase of calmodulin with MLCK using the “zero-length” crossactivity, which is consistent with the cross-linking of linking reagent DCC (Carraway and Koshland, 1972), sepacalmodulin in close proximity to a regulatory region of ration of the reaction products on DEAE-cellulose, and the myosin light chain kinase
MLCK were incubated with DCC, and a major cross-linked tion that was separated onDEAE-cellulose
Summary
The interaction between bovine testes calmodulin 1987; Kennelly et al, 1987; Pearson et al, 1988). The results are measurement of kinase activity of a fraction containing a consistent with a mechanism whereby the association cross-linked MLCK are reported. Time of 18h (Fig.1).Higher molecular weight speciesappeared The cross-linked complex from two other preparations was in low yield as the calmodulin to MLCK ratiowas increased.
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