Purification, cDNA cloning and expression of 15-oxoprostaglandin 13-reductase from pig lung
Purification, cDNA cloning and expression of 15-oxoprostaglandin 13-reductase from pig lung
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DOI：
10.1042/bj3300103
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发表时间：
1998-02-15
影响因子：
4.1
通讯作者：
Tai, HH
中科院分区：
文献类型：
--
作者：
Ensor, CM;Zhang, HX;Tai, HH
15-氧前列腺素13-还原酶（PGR）已从猪肺中纯化至表观均一性。通过SDS/PAGE和非变性PAGE估计该酶具有36 kDa的分子量，表明该酶是单体。发现15-oxo-PGE（1）、15-oxo-PGE（2）和15-oxo-PGF（2 α）是该酶的底物，而相应的15-羟基野牡丹素不是。未观察到逆反应，即13，14-二氢-15-氧代-PGE（1）氧化为15-氧代-PGE（1）。NADH或NADPH均可作为辅酶。然而，具有NADH的V-max约为。3-倍，而NADPH的Km约为。十分之一是NADH。通过PCR和文库筛选获得cDNA克隆。一个600 bp的PCR产物含有三个不同的胰蛋白酶肽的序列，从纯化的PCR产物用于cDNA文库筛选噬斑杂交。获得了含有987 bp的完整PCR编码序列的cDNA克隆。该序列编码329个氨基酸残基的蛋白质，计算分子量为35791 Da，同源性分析表明该序列实际上与白三烯B-4（LTB 4）12-羟基脱氢酶的序列相同[Yokomizo，Ogawa，Uozumi，Kume，Izumi and Shimizu（1996）J.Biol.Chem.271，2844-2850]。该cDNA在大肠杆菌中的表达导致表现出PCR和LTB 4 12-羟基脱氢酶活性的蛋白质。然而，用15-氧代-PGE（1）作为底物的PCR的比活性约为1.00%。300-是LTB 4 12-羟基脱氢酶的两倍。这些结果表明，克隆的cDNA编码的蛋白质具有两种不同的酶活性，与15-oxoeaglandins作为优选的底物。
15-Oxoprostaglandin 13-reductase (PGR) has been purified to apparent homogeneity from pig lung. The enzyme was estimated to have a molecular mass of 36 kDa by both SDS/PAGE and non-denaturing PAGE, indicating that the enzyme is a monomer. 15-Oxo-PGE(1), 15-oxo-PGE(2) and 15-oxo-PGF(2 alpha) were found to be substrates for the enzyme, whereas the corresponding 15-hydroxyprostaglandins were not. The reverse reaction, the oxidation of 13,14-dihydro-15-oxo-PGE(1) to 15-oxo-PGE(1), was not observed. Either NADH or NADPH could serve as a coenzyme. However, the V-max with NADH was approx. 3-fold that with NADPH, while the K-m for NADPH was approx. one-tenth that for NADH. Cloning of the cDNA was achieved by PCR and library screening. A 600 bp PCR product containing the sequences of three different tryptic peptides derived from purified PGR was used for cDNA library screening by plaque hybridization. A cDNA clone that contained the entire PGR coding sequence of 987 bp was obtained. The sequence codes for a protein of 329 amino acid residues with a calculated molecular mass of 35791 Da. Homology analysis indicated that the sequence is virtually identical with that of leukotriene B-4 (LTB4) 12-hydroxydehydrogenase [Yokomizo, Ogawa, Uozumi, Kume, Izumi and Shimizu (1996) J. Biol. Chem. 271, 2844-2850]. Expression of this cDNA in Escherichia coli resulted in a protein exhibiting both PGR and LTB4 12-hydroxydehydrogenase activities. However, the specific activity of PGR with 15-oxo-PGE(1) as a substrate was approx. 300-fold that of LTB4 12-hydroxydehydrogenase. These results indicate that the cloned cDNA codes for a protein with two different enzyme activities, with 15-oxoprostaglandins as the preferred substrate.