19] Purification of PGD2 11-ketoreductase from rabbit liver

Abstract

This chapter describes the purification of prostaglandin (PG)D 2 11-ketoreductase activity from rabbit liver to apparent homogeneity. Thus, PGD 2 -11-ketoreductase may be a major enzymic pathway for the metabolism of endogenous PGD 2 . Various steps for the purification discussed in the chapter are (1) preparation of crude extract, (2) ammonium sulfate fractionation,(3) diethylaminoethyl (DEAE)-cellulose chromatography, (4) isoelectric focusing and gel electrophoresis, (5) radiometric assay of PGD 2 ll-ketoreductase, and (6) platelet aggregation studies. It is found from the yield and specific activity that little activity was lost from the cytoplasmic fractions during ammonium sulfate fractionation. After chromatography on two DEAE columns and isoelectric focusing, approximately 0.2% of the total cytoplasmic protein but 24% of the PGD 2 , 11-ketoreductase activity remained. In the purification procedure, the enzyme is first separated by the DEAE-Sephadex column from other proteins by a step gradient of NaCl in buffer. When the enzyme fraction was further purified by DEAE-cellulose column chromatography, the enzyme activity was eluted with buffer as one single peak. The active enzyme fractions from DEAE-cellulose column were focused using an analytical isoelectric focusing column containing a pH gradient of 3.5 to 10.0. The enzyme activity is found to focus with a fraction having a pH value of 5.8, indicating the isoelectric point (pI) of the enzyme.