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

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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 μg protein. The minimal estimate value of the Vmax for InsP3 5‐phosphatase was in the range 20–35 μmol · min−1· mg protein−1.