Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages
Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages
复制标题
DOI：
10.1006/abbi.2001.2614
复制
发表时间：
2002-01-15
影响因子：
3.9
通讯作者：
Hayaishi, O
中科院分区：
文献类型：
--
作者：
Lazarus, M;Kubata, BK;Hayaishi, O
我们克隆了小鼠微粒体前列腺素（PG） E合成酶-1 （mPGES-1）的cDNA，并在大肠杆菌中进行了表达。含有重组mPGES-1的膜组分在GSH存在下催化PGH(2)异构化为PGE(2)， PGH(2)的k -m值为130 muM， GSH的k -m值为37 muM，周转时间为600 min(-1), k(cat)/ k -m比为4.6 min(-1) muM（-1）。对重组mPGES-1进行纯化，制备高特异性的mPGES-1多克隆抗体。该抗体在脂多糖处理小鼠腹膜巨噬细胞微粒体部分的Western blotting上显示为单带。Northern和Western blotting分析显示，脂多糖处理小鼠巨噬细胞后，mPGES-1与环氧化酶-2一起被诱导。共聚焦免疫荧光显微镜显示mPGES-1和环氧化酶-2在脂多糖处理的巨噬细胞中共定位。综上所述，这些结果表明mPGES-1是在脂多糖处理的活化巨噬细胞中产生PGE(2)的有效下游酶。(C) 2001 Elsevier Science。
We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH(2) to PGE(2) in the presence of GSH with K-m values of 130 muM for PGH(2) and 37 muM for GSH, a turnover number of 600 min(-1), and a k(cat)/K-m ratio of 4.6 min(-1) muM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE(2) in the activated macrophages treated by lipopolysaccharide. (C) 2001 Elsevier Science.