人胎盘谷胱甘肽S-转移酶的纯化和性质

将人胎盘组织粗匀浆经105000×g超速离心后,用S-已基谷胱甘肽-琼脂糖-6B亲和层析一步纯化法获得电泳纯的人胎盘GST(简称GST-π)。其比活性较粗匀浆高194.7倍,回收率为50％。再经DE_(52)交换柱进一步纯化,用KCl浓度梯度洗脱后为单一锐峰,等电聚集电泳呈一条带,等电点(pI)为4.60。GST-π经TSKgel-G3000SW柱高效液相层析,也为单一对称锐峰,测得其分子量为45.2kD;在SDS-PAGE电泳也为单一区带,测得其亚基单位的分子量为22.5kD。GST-π氨基酸组成分析可检出十六种氨基酸,其中以谷氨酸、亮氨酸、丙氨酸、天冬氨酸及甘氨酸含量较高。 GST-π酶动力学研究证明GST-π以GST和CDNB为底物时km值分别为0.16mmol/L和0.55mmol/L,经测定表明,GST-π的最适作用pH值为7.5,在pH6.5—9的范围内较为稳定,体外GST-π在温度超过25℃对容易失活。以GST-π为抗原,得到兔抗人GST-π抗血清,其效价为1:32,与人肝GSTs不发生免疫交叉反应。

Purification and Characterization of the Glutathione S- Transferase From Human Placenta

The glutathione S-transferase (EC 2.5.1.18) of human placenta (GST-π) was purified approximately 194.7 fold with 50% yeild by affinity chromatography on S-hexylglutathione-linked Sepharose-6B column from 105000 × g supernatant of human placental homogenate. In further purification by DEAE-Cellulose column chromatography, the GST-π showed only one band with pI. of 4.6 In TSK-gel-G 3000 SW HPLC chromatography, GST-π showed one sharp peak with Mr of 45kD. The SDS-PAGE electrophoresis revealed that the GST-π is composed of two identical sub-units with Mr of 22.5 kD.Amino acid composition analysis of GST-re revealed that Glu Leu, Asp, Gly and Ala were its major components. Rabbit antiserum raised against human placental GST was prepared and the antiserum titer measured by Ouchterlony double-diffusion experiment was 1:32.