Purification and characterization of membrane-bound prostaglandin E synthase from bovine heart.
Prostaglandin (PG) E synthase was solubilized with 6 mM sodium deoxycholate from the microsomal fraction of bovine hearts. The enzyme was purified by about 800-fold to apparent homogeneity. The specific activity of the purified enzyme was about 830 mU/mg of protein, and the K(m) value for PGH(2) was 24 microM. The molecular weight of the enzyme was about 31000 on SDS-polyacrylamide gel electrophoresis and was about 60000 by gel filtration. The enzyme was separated from glutathione (GSH) S-transferase by DEAE-Toyopearl column chromatography, and did not exhibit any GSH S-transferase activity towards four different substrates. The purified enzyme was active in the absence of GSH, but it was activated by various SH-reducing reagents including dithiothreitol, GSH, or beta-mercaptoethanol. This is the first reported purification of membrane-bound PGE synthase to apparent homogeneity.
Watanabe
K
K
Second Department, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565, Japan. watanabe@po.cc.toua-u.ac.jp
Kurihara
K
K
Suzuki
T
T
D000818:Animals; D002417:Cattle; D003840:Deoxycholic Acid; D003902:Detergents; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology*; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D000071876:Prostaglandin-E Synthases; D012756:Sheep; D012995:Solubility
D003902:Detergents; D003840:Deoxycholic Acid; D008623:Mercaptoethanol; D005982:Glutathione Transferase; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; D005978:Glutathione; D004229:Dithiothreitol