Purification of bovine brain inositol-1,4,5-trisphosphate 5-phosphatase

Abstract

In bovine brain, two soluble inositol-1,4,5-trisphosphate (InsP3) 5-phosphatases, which catalyse the dephosphorylation of InsP3 to inositol 1,4-bisphosphate, have been separated by DEAE-Sephacel. Type I, i.e. the first eluted enzyme, is the main soluble form and is reminiscent of the membrane-bound enzyme by multiple criteria. Type I was purified to apparent homogeneity by a method involving chromatography on DEAE-Sephacel, Blue-Sepharose, Sephacryl S-200, phosphocellulose, and C18 HPLC. A single protein band of 42-43 kDa was identified by SDS/PAGE, corresponding to the peak of maximal activity. InsP3 5-phosphatase was purified to apparent homogeneity to a final yield of 45-50 micrograms protein. The minimal estimate value of the Vmax for InsP3 5-phosphatase was in the range 20-35 mumol.min-1.mg protein-1.