PRODUCTION OF RECOMBINANT HUMAN BRAIN-TYPE-I INOSITOL-1,4,5-TRISPHOSPHATE 5-PHOSPHATASE IN ESCHERICHIA-COLI - LACK OF PHOSPHORYLATION BY PROTEIN-KINASE-C

The dephosphorylation of inositol 1,4,5-trisphosphate (InsP(3)) to inositol 1,4-bisphosphate is catalyzed by InsP(3) 5-phosphatase. The coding region of human brain type I InsP(3) 5-phosphatase was expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The relative molecular mass of the purified fusion protein (MBP-InsP(3)-5-phosphatase) was approximately M(r) 85 000 as analysed by SDS/PAGE. The yield was about 10 mg fusion protein/l lysate. After cleavage from MBP with factor Xa, the specific activity of recombinant 5-phosphatase was 120-250 mu mol . mg(-1). min(-1). The molecular mass of purified protein by SDS/PAGE was M(r) 43 000. The activity was inactivated by p-hydroxymercuribenzoate. The possibility that protein kinase C might phosphorylate InsP(3) 5-phosphatase was tested on the purified 43 000 M(r) protein. In this study, we show that recombinant 5-phosphatase is not a substrate of protein kinase C.