A purification strategy for inositol 1,4,5-trisphosphate 3-kinase from rat liver based upon heparin interaction chromatography. |
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Authors: | A Conigrave A Patwardhan L Broomhead B Roufogalis |
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Institution: | Department of Pharmacy, University of Sydney, N.S.W., Australia. |
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Abstract: | 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. |
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