Yagisawa H, Hirata M, Kanematsu T, Watanabe Y, Ozaki S, Sakuma K, Tanaka H, Yabuta N, Kamata H, Hirata H, Nojima H
It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilizedm atrix was the 6, isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-6, in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(l,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100- kDa protein showed high Ins(1,4,5)P3 binding. Tlyhsea te was also purified to near homogeneity using a glutathi one- Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as theb rain-derived enzyme. PLC-6, consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-6, which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-6,, which contains 6 basic amino acids, binds to an Ins(l,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P, binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-6, is important for the binding of both Ins(l,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.