Yagisawa H, Hirata M, Kanematsu T, Watanabe Y, Ozaki S, Sakuma K, Tanaka H, Yabuta N, Kamata H, Hirata H
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
J Biol Chem. 1994 Aug 5;269(31):20179-88.
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)-immobilized matrix was the delta 1 isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-delta 1 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(1,4,5)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. The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-delta 1 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-delta 1 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-delta 1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.
先前发现，使用固定有肌醇1,4,5 -三磷酸（Ins(1,4,5)P3）的基质从大鼠脑中纯化得到的85 kDa蛋白是磷脂酰肌醇特异性磷脂酶C（PLC）的δ1同工型。我们在大肠杆菌中表达大鼠PLC - δ1作为与谷胱甘肽S -转移酶的融合蛋白，发现细菌裂解物显示出大量的Ins(1,4,5)P3结合。将裂解物应用于固定有Ins(1,4,5)P3的柱色谱，用仅含一种100 kDa蛋白的2 M NaCl溶液洗脱得到的洗脱物显示出高Ins(1,4,5)P3结合活性。裂解物还使用谷胱甘肽 - 琼脂糖4B亲和系统纯化至接近均一。如此纯化的细菌表达酶显示出与脑源性酶基本相同的肌醇磷酸结合特性。PLC - δ1由氨基末端非保守区和同工酶中两个保守性良好的区域组成，分别命名为X和Y，它们被认为构成了该酶的催化核心。通过结合使用该酶的缺失突变体和蛋白水解产物，我们能够在分子中定位Ins(1,4,5)P3结合结构域。从氨基末端缺失223个残基完全消除了结合活性，而缺失X区域仅部分抑制结合，缺失Y区域则不影响结合。表达的PLC - δ1的一种76 kDa蛋白水解产物在氨基末端缺少60个氨基酸，显示出最小的Ins(1,4,5)P3结合活性。一个由14个氨基酸组成的肽，对应于PLC - δ1的第30 - 43位残基，含有6个碱性氨基酸，能与固定有Ins(1,4,5)P3的基质结合。此外，Ins(1,4,5)P3结合被含有磷脂酰肌醇4,5 -二磷酸的磷脂囊泡阻断。综合这些结果表明，PLC - δ1的氨基末端结构域对于Ins(1,4,5)P3和磷脂酰肌醇4,5 -二磷酸的结合都很重要。
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