Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-delta 1.
Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-delta 1.
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磷脂酰肌醇特异性磷脂酶 C-delta 1 的肌醇 1,4,5-三磷酸结合域的表达和表征。
DOI：
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发表时间：
1994
影响因子：
4.8
通讯作者：
Hajime Hirata
中科院分区：
文献类型：
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作者：
Hitoshi Yagisawa;Masato Hirata;T. Kanematsu;Y. Watanabe;S. Ozaki;K. Sakuma;H. Tanaka;N. Yabuta;Hideaki Kamata;Hajime Hirata
以前发现，使用肌醇1，4，5-三磷酸（Ins（1，4，5）P3）-固定化基质从大鼠脑中纯化的85-kDa蛋白是磷脂酰肌醇特异性磷脂酶C（PLC）的δ 1亚型。我们在大肠杆菌中表达了大鼠PLC-δ 1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Ins（1，4，5）P3-固定化柱层析，并且用仅含有100-kDa蛋白的2 M NaCl溶液的层析显示高的Ins（1，4，5）P3结合。还使用谷胱甘肽-琼脂糖4 B亲和系统将裂解物纯化至接近均一。如此纯化的细菌表达的酶显示出与脑源性酶基本相同的磷酸肌醇结合特性。PLC-δ 1由氨基端非保守区和两个在同工酶中高度保守的区域（命名为X和Y）组成，这两个区域被认为构成酶的催化核心。使用缺失突变体和酶的蛋白水解产物的组合，我们能够在分子中定位Ins（1，4，5）P3结合结构域。氨基端223个残基的缺失完全消除了结合活性，而X区的缺失仅部分抑制结合，Y区的缺失不影响结合。在氨基末端缺少60个氨基酸的表达的PLC-δ 1的76-kDa蛋白水解产物显示最小的Ins（1，4，5）P3结合活性。肽由对应于PLC-δ 1的残基30-43的14个氨基酸组成，其含有6个碱性氨基酸，结合于Ins（1，4，5）P3固定化基质。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。这些结果综合起来表明，PLC-δ 1的氨基末端结构域对于Ins（1，4，5）P3和磷脂酰肌醇4，5-二磷酸的结合是重要的。
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.