Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages

The activity of inositol-1,4,5-trisphosphate 3-kinase in the cytosol fraction of guinea pig macrophages was assayed with special reference to the dependence on the free Ca²⁺ concentration. The enzyme activity, as assessed by the production of inositol 1,3,4,5-tetrakisphosphate was reversibly activated by free Ca²⁺ concentrations ranging from 10^-7 to 10^-6m. The calmodulin antagonists, W-7 and chlorpromazine, inhibited the Ca²⁺-activated enzyme activity in a dose-dependent fashion, thereby indicating that calmodulin may be involved in the activation by Ca²⁺. The content of calmodulin in the cytosol fraction (about 2.8 μg/mg of cytosol protein) was markedly reduced to less than 0.03 μg/mg of proteins by subfractionation by ammonium sulfate, followed by an anionexchange chromatography. The subfraction obtained by the chromatography showed no Ca²⁺ dependence in the enzyme activity, while an exogenous addition of calmodulin with 10^-6 m Ca^-6 increased the enzyme activity. The enzyme activity was retained on a calmodulin-affinity column in the presence of Ca²⁺, and was eluted from the column by lowering the free Ca²⁺ concentration by adding ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.