CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF A DOG THYROID CDNA-ENCODING A NOVEL INOSITOL 1,4,5-TRISPHOSPHATE 5-PHOSPHATASE

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP(3)) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP(3). This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKA-KKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP(3) and inositol 1,3,4,5-tetrakisphosphate, with apparent K-m values of 28 and 3 mu M respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP(3) 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP(3) 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.