Identification and characterization of a novel type of membrane-associated prostaglandin E synthase.
Membrane-associated prostaglandin E synthase (mPGE synthase) was previously purified to apparent homogeneity from the microsomal fraction of bovine heart (Watanabe, K., et al., Biochim. Biophys. Acta 1439, 406--414, 1999). The N-terminal 22-amino acid sequence of the purified enzyme was identical to that of the 88th to 109th amino acids deduced from the monkey (AB046026) or human (AK024100) cDNA that encodes a hypothetical protein with unknown function. The primary structure has the consensus region of glutaredoxin and of thioredoxin. We constructed an expression plasmid, using the vector (pTrc-HisA) and the monkey cDNA for the 290-amino-acid polypeptide. The recombinant protein with a M(r) of 33 kDa exhibited PGE synthase activity and was purified to apparent homogeneity by nickel-chelating column chromatography. The V(max) and K(m) values for PGH(2) of the purified recombinant mPGE synthase were about 3.3 mumol/min center dot mg of protein and 28 muM, respectively. The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and beta-mercaptoethanol, in order of decreasing effectiveness. Moreover, the mRNA distribution was high in the heart and brain, but the mRNA was not expressed in the seminal vesicles. These results indicate that the recombinant mPGE synthase is identical to the enzyme purified from the microsomal fraction of bovine heart, and is a novel type of mPGE synthase based on the primary structure, a broad specificity of thiol requirement, and tissue distribution.
Tanikawa
Naomi
N
Special Division for Human Life Technology, Cell Dynamics Research Group, National Institute of AIST, Midorigaoka, Ikeda, Osaka 563-8577, Japan.
Ohmiya
Yoshihiro
Y
Ohkubo
Hiroaki
H
Hashimoto
Katsuyuki
K
Kangawa
Kenji
K
Kojima
Masami
M
Ito
Seiji
S
Watanabe
Kikuko
K
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D000882:Haplorhini; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D000071876:Prostaglandin-E Synthases; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014018:Tissue Distribution
D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; D015232:Dinoprostone