Jung A, Schlegel W, Jackisch R, Friedrich E J, Wendel A, Rückrich M F
Hoppe Seylers Z Physiol Chem. 1975 Jun;356(6):787-98.
15-Hydroxyprostaglandin dehydrogenase was isolated from human term placenta up to a final purification of 380-fold. A spec. act. of 2000 mU/mg of protein was reached. The preparation was not homogeneous as judged by analytical disc electrophoresis. The enzyme could be stored in the presence of 50% glycerol and 10mM 2-mercaptoethanol without any loss of activity for at least one year. A distinct single protein band stained after discontinuous polyacrylamide gel electrophoresis was shown by enzymatic activity staining to correspond to 15-hydroxyprostaglandin dehydrogenase activity. Thus no evidence for the exitstence of isoenzymes was obtained. The protein in the final preparation steps showed neither alcohol dehydrogenase, NAD reductase, nor NADH oxidase activity, nor enzymatic conversion of prostaglandin or 15-oxoprostaglandin in the absence of NAD and NADH. No spontaneous reactions between NAD and prostaglandin or NADH and 15-oxoprostaglandin were detectable in the absence of the enzyme. Ethanol and glycerol slightly inhibited the reaction. Various buffers (Tris/HC1, potassium phosphate, HEPES, and triethanolamine) and salts (ammonium chloride, ammonium sulfate, potassium chloride, and sodium chloride) had different effects on the reaction rate. The pH profile of the reaction shows a plateau between pH 7.0 and 7.8 and a steep maximum at pH 9.5. A linear Arrhenius plot was obtained for the temperature dependence of the reaction from 20 to 37 degrees C. The molar activation enthalpy of the reaction was calculated to be 13.1 kcal/mole. The molecular weight of 15-hydroxyprostaglandin dehydrogenase was estimated to be 32000 -/+ 3000 by gel filtration on Sephadex G-150 in the presence of 10mM mercaptoethanol.
从足月人胎盘中分离出15 - 羟基前列腺素脱氢酶，最终纯化倍数达380倍。蛋白质的比活性达到2000 mU/mg。经分析圆盘电泳判断，该制剂并非均一。该酶可在50%甘油和10 mM 2 - 巯基乙醇存在的情况下保存，至少一年内活性无任何损失。不连续聚丙烯酰胺凝胶电泳后染色呈现的一条明显单一蛋白带，经酶活性染色显示对应15 - 羟基前列腺素脱氢酶活性。因此未获得同工酶存在的证据。最终制备步骤中的蛋白质既无乙醇脱氢酶、NAD还原酶、NADH氧化酶活性，在无NAD和NADH时也无前列腺素或15 - 氧代前列腺素的酶促转化。在无酶的情况下，未检测到NAD与前列腺素或NADH与15 - 氧代前列腺素之间的自发反应。乙醇和甘油轻微抑制该反应。各种缓冲液（Tris/HCl、磷酸钾、HEPES和三乙醇胺）及盐（氯化铵、硫酸铵、氯化钾和氯化钠）对反应速率有不同影响。反应的pH曲线在pH 7.0至7.8之间呈平稳状态，在pH 9.5时达到陡峭的最大值。在20至37摄氏度范围内，反应的温度依赖性呈现线性阿伦尼乌斯图。反应的摩尔活化焓经计算为13.1 kcal/mol。在10 mM巯基乙醇存在的情况下，通过Sephadex G - 150凝胶过滤法估计15 - 羟基前列腺素脱氢酶的分子量为32000 ± 3000。
