Open AccessPurification and Characterization of the phosphatidylinositol‐3,4,5‐trisphosphate Phosphatase in Bovine Thymus
Author(s) -
Kabuyama Yukihito,
Nakatsu Noriyuki,
Homma Yoshimi,
Fukui Yasuhisa
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.0350z.x
Subject(s) - phosphatidylinositol , phosphatase , enzyme , biochemistry , sepharose , dephosphorylation , chemistry , molecular mass , alkaline phosphatase , kinase , specific activity , microbiology and biotechnology , biology
Using phosphatidylinositol 3,4,5‐trisphosphate [PtdIns(3,4,5) P 3 ] prepared from phosphatidylinositol 4,5‐bisphosphate and inositolphospholipid 3‐kinase, we identified in bovine thymus extracts the enzyme activity which catalyzed dephosphorylation of PtdIns(3,4,5) P 3 , to produce phosphatidylinositol bisphosphate. Since bovine thymus exhibited the highest level of activity among tissues screened, we tried to purify this enzyme PtdIns(3,4,5) P 3 phosphatase from bovine thymus. After sequential chromatographies using S‐Sepharose, heparin‐Sepharose, blue Sepharose, and Toyopearl HW55, the enzyme was purified 1875‐fold with a yield of 10%. SDS/PAGE analysis revealed that a 120‐kDa protein band copurified with the enzyme activity. The apparent molecular mass of the active 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 that metabolized PtdIns(3,4,5) P 3 , and the enzyme did not hydrolyze phosphatidylinositol 4,5‐bisphosphate, the enzyme may play a critical role in the inositolphospholipid 3‐kinase signalling.