PURIFICATION, CDNA CLONING AND EXPRESSION OF 15-OXOPROSTAGLANDIN 13-REDUCTASE FROM PIG LUNG

15-Oxoprostaglandin 13-reductase (PGR) has been purified to apparent h omogeneity from pig lung. The enzyme was estimated to have a molecular mass of 36 kDa by both SDS/PAGE and non-denaturing PAGE, indicating t hat the enzyme is a monomer. 15-Oxo-PGE(1), 15-oxo-PGE(2) and 15-oxo-P GF(2 alpha) were found to be substrates for the enzyme, whereas the co rresponding 15-hydroxyprostaglandins were not. The reverse reaction, t he oxidation of 13,14-dihydro-15-oxo-PGE(1) to 15-oxo-PGE(1), was not observed. Either NADH or NADPH could serve as a coenzyme. However, the V-max with NADH was approx. 3-fold that with NADPH, while the K-m for NADPH was approx. one-tenth that for NADH. Cloning of the cDNA was ac hieved by PCR and library screening. A 600 bp PCR product containing t he sequences of three different tryptic peptides derived from purified PGR was used for cDNA library screening by plaque hybridization. A cD NA clone that contained the entire PGR coding sequence of 987 bp was o btained. The sequence codes for a protein of 329 amino acid residues w ith a calculated molecular mass of 35791 Da. Homology analysis indicat ed that the sequence is virtually identical with that of leukotriene B -4 (LTB4) 12-hydroxydehydrogenase [Yokomizo, Ogawa, Uozumi, Kume, Izum i and Shimizu (1996) J. Biol. Chem. 271, 2844-2850]. Expression of thi s cDNA in Escherichia coli resulted in a protein exhibiting both PGR a nd LTB4 12-hydroxydehydrogenase activities. However, the specific acti vity of PGR with 15-oxo-PGE(1) as a substrate was approx. 300-fold tha t of LTB4 12-hydroxydehydrogenase. These results indicate that the clo ned cDNA codes for a protein with two different enzyme activities, wit h 15-oxoprostaglandins as the preferred substrate.