从人胎盘中纯化一种NAD⁺依赖性15-羟基前列腺素脱氢酶。

Purification of an NAD+-dependent 15-hydroxyprostaglandin dehydrogenase from the human placenta.

作者信息

Nagai K, Tsuruta K, Mori N

出版信息

Prostaglandins Leukot Med. 1987 Feb;26(2):123-32. doi: 10.1016/0262-1746(87)90107-7.

Abstract

We purified 15-hydroxyprostaglandin dehydrogenase (PGDH) from the human placenta by gel filtration high performance liquid chromatography (GFHPLC) with spectrophotometric assay for the chromophore of 15-keto PG. Purification steps were as follows; homogenization and centrifugation at 10,000g, for 30 min, ammonium sulfate precipitation, DEAE-cellulose (DE-52), Sephadex G-100 gel filtration, NAD-affinity chromatography (AGNAD type 1), and GFHPLC. The specific activity was increased from 1.5 X 10(-2) unit/mg in the initial supernatant to 1272 X 10(-2) after GFHPLC. PGDH showed a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight determined by GFHPLC and electrophoresis was approximately 50,000 daltons. After 4 months of storage, PGDH in a buffer containing 50% glycerol at -20 degrees C retained 80% of its activity. Michaelis constant for PGE2 was 0.4 microM at pH 7.0 with NAD as a cofactor. These properties of PGDH were compared with the results reported in other literature.

摘要

我们通过凝胶过滤高效液相色谱法（GFHPLC），利用对15-酮基前列腺素发色团的分光光度测定法，从人胎盘中纯化出15-羟基前列腺素脱氢酶（PGDH）。纯化步骤如下：匀浆并在10,000g下离心30分钟，硫酸铵沉淀，DEAE-纤维素（DE-52），葡聚糖凝胶G-100凝胶过滤，NAD亲和色谱法（AGNAD 1型）以及GFHPLC。比活性从初始上清液中的1.5×10⁻²单位/毫克增加到GFHPLC后的1272×10⁻²单位/毫克。PGDH在SDS-聚丙烯酰胺凝胶电泳上显示为单一条带，通过GFHPLC和电泳测定的分子量约为50,000道尔顿。在-20℃下于含有50%甘油的缓冲液中储存4个月后，PGDH保留了80%的活性。以NAD作为辅因子时，在pH 7.0条件下，PGDH对PGE2的米氏常数为0.4微摩尔。将PGDH的这些特性与其他文献报道的结果进行了比较。