PRODUCTION OF RECOMBINANT HUMAN BRAIN-TYPE-I INOSITOL-1,4,5-TRISPHOSPHATE 5-PHOSPHATASE IN ESCHERICHIA-COLI - LACK OF PHOSPHORYLATION BY PROTEIN-KINASE-C
C. Erneux et al., PRODUCTION OF RECOMBINANT HUMAN BRAIN-TYPE-I INOSITOL-1,4,5-TRISPHOSPHATE 5-PHOSPHATASE IN ESCHERICHIA-COLI - LACK OF PHOSPHORYLATION BY PROTEIN-KINASE-C, European journal of biochemistry, 234(2), 1995, pp. 598-602
The dephosphorylation of inositol 1,4,5-trisphosphate (InsP(3)) to ino
sitol 1,4-bisphosphate is catalyzed by InsP(3) 5-phosphatase. The codi
ng region of human brain type I InsP(3) 5-phosphatase was expressed as
a fusion protein with the maltose-binding protein (MBP) in Escherichi
a 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). mi
n(-1). The molecular mass of purified protein by SDS/PAGE was M(r) 43
000. The activity was inactivated by p-hydroxymercuribenzoate. The pos
sibility that protein kinase C might phosphorylate InsP(3) 5-phosphata
se was tested on the purified 43 000 M(r) protein. In this study, we s
how that recombinant 5-phosphatase is not a substrate of protein kinas
e C.