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Abstract
Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.
Highlights
of molecularand catalytic propertiesof PGD synthetases purified from rat spleenand brain
This enzymewas more active with PGH
inactive with other synthceotmicpoundsused as the substratesfor alkyl
Summary
The spleenPGD synthetase isreadily inactivated by the isomerasereaction of PGH,to PGD,. The GSH S-transferase reaction continliuneadrly up to 15 min under our standard assayconditions, during which time the turnover numbeorf the transferase reaction catalyzedby this enzyme (70 min-l) is almost comparable with that of the PGD synthetase reactionfor 1 min (1250 rnin") (Table IV). Free form N-terminal amino acid of the brain PGD synthetase was not detectedby dansylation analysis.N terminus of this enzyme is thought to be blocked. The brain PGD synthetasedoesnot absolutely require GSH for the reaction.
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