Purification and characterization of rat brain prostaglandin D synthetase.
Prostaglandin D synthetase was purified 2,600-fold from rat brain to apparent homogeneity, as judged by polyacrylamide gel electrophoresis and ultracentrifugation. The purified enzyme was a monomeric protein with a molecular weight of 27,000 +/- 1,000. The pI value, sedimentation coefficient, and partial specific volume were 4.6, 4.1 s, and 0.73 ml/g, respectively. The enzyme was stable between pH 4 and 11 at the temperature lower than 25 degrees C and resistant to a heat treatment under alkaline conditions (pH 8-11). About 50% of the activity was detected after a heat treatment at 100 degrees C for 5 min at pH 10. However, the enzyme was readily inactivated by the isomerase reaction of prostaglandin H2 to prostaglandin D2. The enzyme required sulfhydryl compounds such as dithiothreitol, glutathione, beta-mercaptoethanol, cysteine, and cysteamine for the reaction, but stoichiometric oxidation of these sulfhydryl compounds was not observed. The optimum pH, Km value for prostaglandin H2, and the turnover number were 9.5, 14 microM, and 170 min-1, respectively. The antibody was raised against the purified enzyme in a rabbit, which showed only one positive band in immunoblotting after gel electrophoresis of crude extracts of the brain at the same position as that of the purified enzyme. More than 90% of the prostaglandin D synthetase activity in the brain was absorbed by an excess amount of the antibody, indicating that our preparation is a major component of the enzyme responsible for the biosynthesis of prostaglandin D2 in the brain.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007163:Immunosorbent Techniques; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D015230:Prostaglandin D2; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism; D044262:Prostaglandin H2; D011451:Prostaglandin-Endoperoxide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D011457:Prostaglandins D / Q000378:metabolism; D011463:Prostaglandins H / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014461:Ultracentrifugation
D011450:Prostaglandin Endoperoxides, Synthetic; D011457:Prostaglandins D; D011463:Prostaglandins H; D013438:Sulfhydryl Compounds; D044262:Prostaglandin H2; D011451:Prostaglandin-Endoperoxide Synthases; D015230:Prostaglandin D2