Calmodulin activates inositol 1,4,5-trisphosphate 3-kinase activity in pig aortic smooth muscle.
The effects of calmodulin (CaM) on inositol 1,4,5-trisphosphate (InsP3) 3-kinase activity in pig aortic smooth muscle were examined. The cytosol fraction of muscle cells, containing 1.2-2.0 micrograms of CaM/mg of cytosol protein (thus 0.12-0.2%, w/w), showed a Ca2+-dependent InsP3 3-kinase activity, and there was no further activation by exogenous addition of CaM purified from dog brain. (NH4)2SO4 fractionation of the cytosol fraction revealed that a 20-60%-satd.-(NH4)2SO4 fraction was rich in the enzyme activity, and the activity without exogenous CaM was still dependent on Ca2+, although the CaM content in this fraction was minute (0.013-0.016%, w/w). The kinase activity observed in the absence of exogenous CaM became insensitive to Ca2+ when a 20-60%-satd.-(NH4)2SO4 fraction was applied to a DEAE-cellulose column, but exogenous addition of CaM increased the enzyme activity from 80-120 to 450 pmol/min per mg of protein, with addition of 10 microM free Ca2+. A fraction separated by DEAE-cellulose chromatography was applied to a CaM affinity column. The kinase activity was retained on the column in the presence of Ca2+, and was eluted by lowering the free Ca2+ concentration by adding EGTA. These results directly show that CaM activates InsP3 3-kinase activity and the enzyme becomes sensitive to Ca2+.
Yamaguchi
K
K
Department of Biochemistry, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Hirata
M
M
Kuriyama
H
H
D000818:Animals; D002147:Calmodulin / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013552:Swine
D002147:Calmodulin; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase