A purification strategy for inositol 1,4,5-trisphosphate 3-kinase from rat liver based upon heparin interaction chromatography.
Rat liver inositol 1,4,5-trisphosphate [Ins (1,4,5)P3] 3-kinase was purified in high yield by a three-step procedure reliant upon chromatography on heparin and calmodulin agarose. Purified enzyme was stable in the presence of the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) (0.1-0.5%) and the sulphydryl reducing reagent dithiothreitol (DTT). The purified enzyme was activated 2-3-fold by Ca2+ (1 microM) in the presence of calmodulin. Pyrophosphate and heparin were identified as inhibitors of the enzyme.
Conigrave
A
A
Department of Pharmacy, University of Sydney, N.S.W., Australia.
Patwardhan
A
A
Broomhead
L
L
Roufogalis
B
B
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D006493:Heparin / Q000737:chemistry; D007295:Inositol Phosphates / Q000096:biosynthesis; D008099:Liver / Q000201:enzymology*; D008297:Male; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D011919:Rats, Inbred Strains; D012685:Sepharose
D002147:Calmodulin; D007295:Inositol Phosphates; C081655:inositol-1,3,4,5-tetrakisphosphate; D006493:Heparin; D012685:Sepharose; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium