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Characterization of Highly Functional Phospholipase D Produced by Streptoverticillium cinnamoneum

肉桂链轮丝菌产生的高功能磷脂酶 D 的表征

基本信息

批准号：
10650786
负责人：
FUKUDA Hideki
金额：
$ 2.18万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Phospholipase D (PLD1), secreted into the culture medium of Streptoverticillium cinnnamoneum, has been purified to homogeneity and characterized. The Stv.PLD efficiently catalyzes both hydrolysis and transphosphatidylation of various phospholipids, including phosphatidylethanolamine (PE), phosphatidylcholinw (PC), and phosphatidylserine (PS). However, the substrate specificity differs between the two reactions ; PE serves as the most preferred substrate for the hydrolysis, but PC and PS are better substrates than PE for the transphosphatidylation. In addition, the transphosphatidylation but not the hydrolysis of PE and PC is markedly activated on the addition of metal ions, especially ALィイD13ィエD1ィイD2+ィエD2. Nucleotide and amino acid sequence determination of the Stv.PLD revealed the presence of common structural motifs identified in all PLD sequences from various species.The PLD located in the membrane fraction of Stv.(PLD2) is about 35-40-kDa-monomer enzyme, which is different from the secreted into the culture medium and the smallest molecule among the known PLDs. Anti-PLD1 polyclonal antibody did not cross react with PLD2 by Western blotting, suggesting that the overall structure of PLD2 is not similar to that of PLD1. PLD2 preferentially hydrolyzes PE over PC as a substrate. In the presence of ethanol as a donor of polar headgroup, PLD2 could not catalyze the transphosphatidylation reaction and exclusively produced phosphatidic acid. Thus, The enzymatic properties of PLD2 appear to be substantially different from those of PLD1.

磷脂酶D（PLD 1），分泌到培养基中的Streptoverticillium cinnnamoneum，已被纯化到同质和表征。该Stv.PLD有效地催化各种磷脂的水解和转磷脂酰化，包括磷脂酰乙醇胺（PE）、磷脂酰胆碱（PC）和磷脂酰丝氨酸（PS）。然而，两种反应的底物特异性不同; PE作为水解的最优选底物，但PC和PS是比PE更好的转磷脂酰化底物。此外，在添加金属离子时，特别是AL β D13 β D1 β D2+ β D2，PE和PC的转磷脂酰化而不是水解被显著激活。核苷酸和氨基酸序列分析表明，不同种属的PLD序列中存在共同的结构基序，PLD定位于Stv的膜部分。PLD 2是一种分子量约为35-40 kDa的单体酶，与分泌到培养基中的酶不同，是已知PLD中分子量最小的。Western blotting结果显示，抗PLD 1多克隆抗体与PLD 2无交叉反应，说明PLD 2的整体结构与PLD 1不同。PLD 2优先水解PE而不是PC作为底物。在乙醇作为极性头基供体的情况下，PLD 2不能催化转磷脂酰化反应，只能产生磷脂酸。因此，PLD 2的酶性质似乎与PLD 1的酶性质基本上不同。