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(-6)M. 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 micrograms/mg of cytosol protein) was markedly reduced to less than 0.03 microgram/mg of proteins by subfractionation by ammonium sulfate, followed by an anion-exchange 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 Ca2+ 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(beta-aminoethyl ether)-N,N'-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.
Kimura
Y
Y
Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Hirata
M
M
Yamaguchi
K
K
Koga
T
T
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000502:physiology*; D002746:Chlorpromazine / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006168:Guinea Pigs; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013449:Sulfonamides / Q000494:pharmacology
D002147:Calmodulin; D013449:Sulfonamides; C017967:W 7; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium; D002746:Chlorpromazine