Purification of Nad+-Dependent 15-Hydroxyprostaglandin Dehydrogenase from Porcine Kidney

The cytoplasmic NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase (11α, 15-dihydroxy-9-oxoprost-13-enoate: NAD⁺ 15-oxidoreductase, EC 1.1.1.141) from porcine kidney was purified to homogeneity by a series of chromatographic techniques including DEAE-Sepharose CL-6B column chromatography, Blue Sepharose CL-6B column chromatography, Sephadex G-100 chromatography and Mono-P isoelectrofocusing column chromatography. The enzyme was eluted at pH 5.80 on isoelectrofocusing column, indicating that the porcine renal enzyme is an acidic protein. A single band with 32 K was observed in SDS-gel electrophoretic analysis. A monoclonal antibody against human placental enzyme was used to confirm the porcine renal enzyme. The 32 K protein of purified porcine renal enzyme in SDS-polyacrylamide gel cross-reacted with the monoclonal antibody in a Western blot analysis. The amino acid composition of porcine renal enzyme was also analyzed. Nine tyrosine molecules were observed in the subunit (32 K) of porcine renal enzyme, while no tyrosine has been reported in human placental enzyme.