Molecular characterization of a novel type of prostamide/prostaglandin F synthase, belonging to the thioredoxin-like superfamily
Molecular characterization of a novel type of prostamide/prostaglandin F synthase, belonging to the thioredoxin-like superfamily
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DOI：
10.1074/jbc.m705638200
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发表时间：
2008-01-11
影响因子：
4.8
通讯作者：
Watanabe, Kikuko
中科院分区：
文献类型：
--
作者：
Moriuchi, Hiroshi;Koda, Noriko;Watanabe, Kikuko
在小鼠和猪脑中发现了前列腺素F（PGF）乙醇酰胺（前列腺酰胺F）合酶，其催化前列腺酰胺H-2还原为前列腺酰胺F-2 α，F-。从猪脑中纯化了该酶，并测定了其氨基酸序列。小鼠酶由603 bp开放阅读框组成，编码分子量为21，669的201个氨基酸的多肽。氨基酸序列将该酶置于硫氧还蛋白样超家族中，其中Cys（44）是活性位点。在大肠杆菌中表达的酶以及天然酶不仅催化前列腺酰胺H-2还原为前列腺酰胺F-2 α，而且催化PGH（2）还原为PGF（2 α）。前列腺酰胺H-2的Vmax和Km值分别约为0.25 μ mol/min中心点mg蛋白质和7.6 μ M，PGH（2）的Vmax和Km值分别约为0.69 μ mol/min中心点mg蛋白质和6.9 μ M。PGE（2）和PGD（2）都不是这种合成酶的底物。基于这些数据，我们将酶命名为前列腺酰胺/PGF合酶。虽然该酶对还原剂表现出广泛的特异性，但还原硫氧还蛋白优先充当该酶的还原当量供体。北方和Western印迹分析表明，该酶主要分布在脑和脊髓中，脊髓中的免疫组织化学研究表明，该酶主要存在于胞浆中。这些结果表明，前列腺酰胺/PGF合成酶可能在中枢神经系统中发挥重要的功能作用。
Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H-2 to prostamide F-2 alpha,F-, was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669. The amino acid sequence placed the enzyme in the thioredoxin-like superfamily with Cys(44) being the active site. The enzyme expressed in Escherichia coli as well as the native enzyme catalyzed not only the reduction of prostamide H-2 to prostamide F-2 alpha but also that of PGH(2) to PGF(2 alpha). The V-max and Km values for prostamide H-2 were about 0.25 mu mol/min center dot mg of protein and 7.6 mu M, respectively, and those for PGH(2) were about 0.69 mu mol/min center dot mg of protein and 6.9 mu M, respectively. Neither PGE(2) nor PGD(2) served as a substrate for this synthase. Based on these data, we named the enzyme prostamide/PGF synthase. Although the enzyme showed a broad specificity for reductants, reduced thioredoxin preferentially served as a reducing equivalent donor for this enzyme. Moreover, Northern and Western blot analyses in addition to the prostamide F synthase activity showed that the enzyme was mainly distributed in the brain and spinal cord, and the immunohistochemical study in the spinal cord showed that the enzyme was found mainly in the cytosol. These results suggest that prostamide/PGF synthase may play an important functional role in the central nervous system.