PURIFICATION AND CHARACTERIZATION OF THE PHOSPHATIDYLINOSITOL-3,4,5-TRISPHOSPHATE PHOSPHATASE IN BOVINE THYMUS

Using phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P-3] prep ared from phosphatidylinositol 4-5-bisphosphate and inositolphospholip id 3-kinase, we identified in bovine thymus extracts the enzyme activi ty which catalyzed dephosphorylation of PtdIns(3,3,5)P-3, to produce p hosphatidylinosirol bisphosphate. Since bovine thymus exhibited the hi ghest level of activity among tissues screened, we tried to purify thi s enzyme PtdIns(3,4,5)P-3 phosphatase from bovine thymus. After sequen tial chromatographies using S-Sepharose, heparin-Sepharose, blue Sepha rose, and Toyopearl HW55, the enzyme was purified 1875-fold with a yie ld of 10%. SDS/PAGE analysis revealed that a 120-kDa protein band copu rified with the enzyme activity. The apparent molecular mass of the ac tive protein was 120 kDa on size-exclusion chromatography, suggesting that the 120-kDa band on SDS/PAGE is the PtdIns(3,4,5)P-3 phosphatase. Since PtdIns(3,4,5)P-3 phosphatase seemed to be the only activity tha t metabolized PtdIns(3,3,5)P-3, and the enzyme did not hydrolyze phosp hatidylinositol 4,5-bisphosphate, the enzyme may play a critical role in the inositolphospholipid 3-kinase signalling.