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 (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.
Interdisciplinary Research Institute (IRIBHN), Brussels, Belgium.
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Erneux
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De Smedt
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Moreau
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Rider
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D000818:Animals; D001921:Brain / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D000072181:Inositol Polyphosphate 5-Phosphatases; D008297:Male; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000494:pharmacology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis*
D007106:Immune Sera; D011994:Recombinant Proteins; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases