Purification and properties of prostaglandin 9‐ketoreductase from pig and human kidney

Abstract

Prostaglandin 9‐ketoreductase (PG‐9‐KR) was purified from pig kidney to homogeneity, as judged by SDS/PAGE using an improved procedure. The enzyme is pro‐ S stereoselective with regard to hydrogen transfer from NADPH with prostaglandin E 2 as substrate and reduces its 9‐keto group with approximately 90% stereoselectivity to form prostaglandin F 2α . Approximately 8% of the prostaglandin F formed has the β‐configuration. In addition to catalyzing the interconversion of prostaglandin E 2 to F 2α , PG‐9‐KR also oxidizes prostaglandin E 2 , F 2α and D 2 to their corresponding, biologically inactive, 15‐keto metabolites. Incubation of PG‐9‐KR with prostaglandin F 2α and NAD + leads to the preferential formation of 15‐keto prostaglandin F 2α rather than prostaglandin E 2 . This suggests that the prostaglandin E 2 /prostaglandin F 2α ratio is not determined by the NADP + /NADPH redox couple. The enzyme also reduces various other carbonyl compounds (e.g. 9,10‐phen‐anthrenequinone) with high efficiency. The catalytic properties measured for PG‐9‐KR suggest that its in vivo function is unlikely to be to catalyze formation of prostaglandin F 2α . The monomeric enzyme has a molecular mass of 32 kDa and exists as four isoforms, as judged by isoelectric focusing. PG‐9‐KR contains 1.9 mol Zn 2+ /mol enzyme and no other cofactors. Human kidney PG‐9‐KR was also purified to homogeneity. The human enzyme has a molecular mass of 34 kDa and also exists as four isoforms. Polyclonal antibodies raised against pig kidney PG‐9‐KR cross‐react with human kidney PG‐9‐KR and also with human brain carbonyl reductase, as demonstrated by Western blot analysis. Sequence data of tryptic peptides from pig kidney PG‐9‐KR show >90% identity with human placenta carbonyl reductase. From comparison of several properties (catalytical, structural and immunological properties), it is concluded that PG‐9‐KR and carbonyl reductase are identical enzymes.