Regular Article
Identification and Characterization of a Novel Type of Membrane-Associated Prostaglandin E Synthase

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Abstract

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 Mr of 33 kDa exhibited PGE synthase activity and was purified to apparent homogeneity by nickel-chelating column chromatography. The Vmax and Km values for PGH2 of the purified recombinant mPGE synthase were about 3.3 μmol/min · mg of protein and 28 μM, respectively. The recombinant enzyme was activated by various SH-reducing reagents, i.e., dithiothreitol, glutathione (GSH), and β-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.

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Abbreviations used: PG, prostaglandin; mPGE synthase, membrane-associated prostaglandin E synthase; GSH, glutathione; KPB, potassium phosphate buffer; DTT, dithiothreitol; Mer, 2-mercaptoethanol; COX, cyclooxygenase.

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To whom correspondence should be addressed at Division of Life Science, Graduate School of Integrated Science and Art, University of East Asia, 2-1 Ichinomiya-gakuencho, Shimonoseki, Yamaguchi 751-8503, Japan. Fax: 0832-57-5152. E-mail: [email protected].

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