PURIFICATION AND PROPERTIES OF PROSTAGLANDIN 9-KETOREDUCTASE FROM PIG AND HUMAN KIDNEY - IDENTITY WITH HUMAN CARBONYL REDUCTASE

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 E2 as substrate and reduces its 9-keto group with approximately 90% stereoselectivity to form prostaglandin F2-alpha. Approximately 8% of the prostaglandin F formed has the beta-configuration. In addition to catalyzing the interconversion of prostaglandin E2 to F2-alpha, PG-9-KR also oxidizes prostaglandin E2, F2-alpha and D2 to their corresponding, biologically inactive, 15-keto metabolites. Incubation of PG-9-KR with prostaglandin F2-alpha and NAD+ leads to the preferential formation of 15-keto prostaglandin F2-alpha rather than prostaglandin E2. This suggests that the prostaglandin E2/prostaglandin F2-alpha ratio is not determined by the NADP+/NADPH redox couple. The enzyme also reduces various other carbonyl compounds (e.g. 9,10-phenanthrenequinone) 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 F2-alpha. 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 Zn2+/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.