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Production of recombinant human brain type I inositol-1,4,5-trisphosphate 5-phosphatase in Escherichia coli: lack of phosphorylation by protein kinase C

par Erneux, Christophe ;De Smedt, Florence ;Moreau, Colette ;Rider, Mark H.;Communi, David
Référence European journal of biochemistry / FEBS, 234, 2, page (598-602)
Publication Publié, 1995

Article révisé par les pairs

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Résumé :

The dephosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,4-bisphosphate is catalyzed by InsP3 5-phosphatase. The coding region of human brain type I InsP3 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-InsP3-5-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 mumol.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 InsP3 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.