Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.
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 PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-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 PGE2 in the activated macrophages treated by lipopolysaccharide.
Lazarus
Michael
M
Department of Molecular Behavioral Biology, Japan Science and Technology Corporation, Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan.
Kubata
Bruno Kilunga
BK
Eguchi
Naomi
N
Fujitani
Yasushi
Y
Urade
Yoshihiro
Y
Hayaishi
Osamu
O
D000818:Animals; D003001:Cloning, Molecular; D051546:Cyclooxygenase 2; D006168:Guinea Pigs; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000302:isolation & purification*; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D000071876:Prostaglandin-E Synthases; D011451:Prostaglandin-Endoperoxide Synthases / Q000302:isolation & purification*
D007527:Isoenzymes; D008070:Lipopolysaccharides; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; C000605865:Ptges protein, mouse