Purification and characterization of inositol 1,4,5-trisphosphate 3-kinase from pig aortic smooth muscle.
Inositol 1,4,5-trisphosphate (InsP3) 3-kinase, which phosphorylates InsP3 to form inositol 1,3,4,5-tetrakisphosphate, was purified to apparent homogeneity by (NH4)2SO4 fractionation and sequential chromatographic steps on DEAE-sepharose, calmodulin-Affi-Gel and DEAE-5PW h.p.l.c. The purified enzyme had a specific activity of 24.4 nmol of inositol tetrakisphosphate formed/min per mg of protein, which represented a purification of approx. 195-fold with a 0.29% recovery, compared with the cytosol fraction of the muscle. SDS/polyacrylamide-gel electrophoresis showed a single protein-staining band of Mr 93,000. Moreover, the major protein peak, of Mr 84,000, was detected by TSK gel G3000SW gel-permeation chromatography of the purified sample. As this value was approximately consistent with the Mr determined by SDS/polyacrylamide-gel-electrophoretic analysis, the InsP3 3-kinase might be a monomeric enzyme. The purified enzyme had a Km for InsP3 of 0.4 microM, with an optimum pH range of 5.8-7.7. The enzyme was maximally activated by calmodulin, with a stoichiometry of 1:1.
Yamaguchi
K
K
Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Hirata
M
M
Kuriyama
H
H
D000818:Animals; D001011:Aorta / Q000201:enzymology; D002147:Calmodulin / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D013552:Swine
D002147:Calmodulin; D007295:Inositol Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase