Identification and characterization of a novel type of membrane-associated prostaglandin E synthase
Identification and characterization of a novel type of membrane-associated prostaglandin E synthase
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DOI：
10.1006/bbrc.2002.6531
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发表时间：
2002-03-08
影响因子：
3.1
通讯作者：
Watanabe, K
中科院分区：
文献类型：
--
作者：
Tanikawa, N;Ohmiya, Y;Watanabe, K
膜相关前列腺素E合酶（mPGE合酶）先前从牛心脏的微粒体部分纯化至表观均一性（Watanabe，K.，等人，Biochim. Biophys. Acta 1439，406414，1999）。纯化酶的N-末端22个氨基酸序列与从编码未知功能的假设蛋白质的猴（AB 046026）或人（AR 024100）cDNA推导的第88至109个氨基酸序列相同。一级结构具有谷氧还蛋白和硫氧还蛋白的共有区。我们使用载体（pTrc-HisA）和猴cDNA构建了290个氨基酸多肽的表达质粒。重组蛋白具有33 kDa的M-r表现出PGE合酶活性，并通过镍螯合柱层析纯化至表观均一性。纯化的重组mPGE合酶的PGH（2）的Vmax和Km值约为3.3 μ mol/min。mg蛋白和28 μ M。重组酶被各种SH-还原试剂激活，即，二硫苏糖醇、谷胱甘肽（GSH）和β-巯基乙醇，以有效性递减的顺序。此外，该mRNA在心脏和大脑中的分布较高，但在精囊中不表达。这些结果表明，重组mPGE合酶是相同的酶纯化从微粒体部分的牛心脏，是一种新型的mPGE合酶的基础上的一级结构，广泛的巯基需求的特异性，和组织分布。(C)2002 Elsevier Science（美国）。
Membrane-associated prostaglandin E synthase (mPGE synthase) was previously purified to apparent homogeneity from the microsomal fraction of bovine heart (Watanabe, K., et aL, Biochim. Biophys. Acta 1439,406414, 1999). The N-terminal 22-amino acid sequence of the purified enzyme was identical to that of the 88th to 109th amino acids deduced from the monkey (AB046026) or human (AR024100) cDNA that encodes a hypothetical protein with unknown function. The primary structure has the consensus region of glutaredoxin and of thioredoxin. We constructed an expression plasmid, using the vector (pTrc-HisA) and the monkey cDNA for the 290-amino-acid polypeptide. The recombinant protein with a M-r of 33 kDa exhibited PGE synthase activity and was purified to apparent homogeneity by nickel-chelating column chromatography. The V-max and K-m values for PGH(2) of the purified recombinant mPGE synthase were about 3.3 mumol/min . mg of protein and 28 muM, respectively. The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and beta-mercaptoethanol, in order of decreasing effectiveness. Moreover, the mRNA distribution was high in the heart and brain, but the mRNA was not expressed in the seminal vesicles. These results indicate that the recombinant mPGE synthase is identical to the enzyme purified from the microsomal fraction of bovine heart, and is a novel type of mPGE synthase based on the primary structure, a broad specificity of thiol requirement, and tissue distribution. (C) 2002 Elsevier Science (USA).