Identification of the bovine brain Ins(1,4,5)P₃ 5‐phosphatase after SDS‐polyacrylamide gel electrophoresis

Abstract

Ins(1,4,5)P a 5-phosphatase catalyzes the dephosphorylation of Ins(l,4,5)P 3 in the 5-position. In a high speed soluble fraction of bovine brain, there are two soluble 5-phosphatases: type I and type II. The purified Ins(l,4,5)P 3 5-phosphatase type I exhibits a major silver-stained band of 43 kDa on denaturing (SDS) gels. It is possible to extract the 5-phosphatase activity form a duplicate lane after gel electrophoresis. The 43 kDa region contains the extractable Ins(1,4,5)P a 5-phosphatase activity.Ca2+; Inositol 1,4,5-trisphosphate 5-phosphatase; (Bovine brain)

Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase

“…A putative initiation codon may direct the synthesis of an InsP3 5-phosphatase of molecular mass 47681 Da, consistent with the molecular mass of the native bovine brain enzyme on an SDS gel, which migrates as a 43 kDa protein (Lemos et al, 1989;Verjans et al, 1992). Although the microsequences were obtained starting with a soluble enzyme, we do not exclude the possibility that clone ETC may encode a membrane-associated protein or that the protein may be anchored to the membrane.…”

“…Analysis of the purified protein by SDS/PAGE and silver staining resulted in a single protein band with a relative molecular mass of 43 kDa (Verjans et al, 1992). Moreover, InsP3 5-phosphatase could be identified by regeneration of activity after electrophoresis (Lemos et al, 1989). Amino acid sequences of the protein were obtained after in situ digestion ofthe whole preparation (about 50 jag of protein); the protein was purified by electrophoresis and electroblotted on a Problot membrane.…”

Purification of bovine brain inositol‐1,4,5‐trisphosphate 5‐phosphatase

“…On SDS/PAGE, both enzyme activities were associated with a 43-kDa proteins, as determined by regeneration of activity (Lemos et al, 1989;Hollande et al, 1991), suggesting that in brain, most InsPJ 5-phosphatase is a Type-I enzyme. In order to obtain sufficient material to generate microsequences, InsP3 5-phosphatase was purified to apparent homogeneity with a final yield of about 45 -50 pg protein.…”