Krook M, Marekov L, Jörnvall H
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Biochemistry. 1990 Jan 23;29(3):738-43. doi: 10.1021/bi00455a021.
Human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase was purified to homogeneity according to a five-step method, with chromatography on DEAE-Sepharose, Blue Sepharose, and Mono-Q FPLC as principal steps. Final yield was 23% and purification about 13,000-fold, with a specific activity of 24,000 milliunits/mg. The subunit molecular weight is about 29,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the native protein molecular weight is about 54,000 as estimated by Sephadex G-100 chromatography, establishing the enzyme to be a dimer of similar-sized protein chains. The subunit N-terminal residue is methionine, and the alpha-amino group is free. The complete primary structure was determined by peptide analysis, based essentially on four different proteolytic treatments (Lys-specific protease, Glu-specific protease, Asp-specific protease, and CNBr). The protein chain is composed of 266 residues, with C-terminal glutamine. A microheterogeneity was detected at position 217, with both Cys and Tyr, in about equal amounts, from a preparation starting with a single placenta. No other subunit heterogeneities were detected. The protein is clearly but distantly related to insect alcohol dehydrogenases, characterized bacterial dehydrogenases of sugar metabolism, and bacterial and eukaryotic steroid dehydrogenases. Together, these results establish that placental 15-hydroxyprostaglandin dehydrogenase is a member of the short-chain nonmetalloenzyme alcohol dehydrogenase protein family. The protein has four cysteine residues (five with the positional microheterogeneity), but there is no evidence for functional importance of any of these residues.(ABSTRACT TRUNCATED AT 250 WORDS)
人胎盘NAD(+)连接的15-羟基前列腺素脱氢酶按照五步方法纯化至同质，主要步骤包括在DEAE-琼脂糖、蓝色琼脂糖和Mono-Q FPLC上进行层析。最终产率为23%，纯化倍数约为13000倍，比活性为24000毫单位/毫克。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳测定，亚基分子量约为29000，通过Sephadex G-100层析估计天然蛋白分子量约为54000，确定该酶为大小相似的蛋白质链的二聚体。亚基N端残基为甲硫氨酸，α-氨基游离。完整的一级结构通过肽分析确定，主要基于四种不同的蛋白水解处理（赖氨酸特异性蛋白酶、谷氨酸特异性蛋白酶、天冬氨酸特异性蛋白酶和溴化氰）。蛋白质链由266个残基组成，C端为谷氨酰胺。从单个胎盘开始制备，在第217位检测到微异质性，同时存在半胱氨酸和酪氨酸，含量大致相等。未检测到其他亚基异质性。该蛋白与昆虫醇脱氢酶、具有特征性的糖代谢细菌脱氢酶以及细菌和真核类固醇脱氢酶有明显但较远的关系。这些结果共同表明，胎盘15-羟基前列腺素脱氢酶是短链非金属酶醇脱氢酶蛋白家族的成员。该蛋白有四个半胱氨酸残基（五个存在位置微异质性），但没有证据表明这些残基中的任何一个具有功能重要性。（摘要截断于250字）
