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 greater than 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.
Schieber
A
A
University of Konstanz, Faculty of Biology, Federal Republic of Germany.
Frank
R W
RW
Ghisla
S
S
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D011453:Prostaglandins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013054:Spectrophotometry, Atomic; D013379:Substrate Specificity; D013552:Swine
D000596:Amino Acids; D011453:Prostaglandins; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D006911:Hydroxyprostaglandin Dehydrogenases; C035858:prostaglandin-E2 9-reductase; D000449:Aldehyde Reductase