An NADP-linked 15-hydroxyprostaglandin dehydrogenase specific for prostaglandin D2 from swine brain.
NADP-linked prostaglandin D2 dehydrogenase was purified from swine brain to apparent homogeneity, as judged by polyacrylamide gel electrophoresis and ultracentrifugation. Using the purified enzyme free of the 15-ketoprostaglandin delta 13-reductase activity, the reaction product was identified as 15-ketoprostaglandin D2 by mass spectrometry and spectrophotometry. The purified enzyme was a monomeric protein of a molecular weight of 29,000 +/- 1,000. It had a relative abundance of hydrophobic amino acids such as leucine, alanine, and valine. The optimal pH of the enzyme reaction was 9.5. The purified enzyme exhibited high substrate specificity for prostaglandin D2 and strict specificity for NADP. The Vmax and the Km values for prostaglandin D2 and NADP were 22.0 nmol/min/mg of protein at 24 degrees C and 52 microM and 0.4 microM, respectively. The Vmax and the Km values for various types of prostaglandins for 8.6 nmol/min/mg of protein at 24 degrees C and 250 microM for D1; 8.0 and 350 for E2; 3.4 and 400 for F2 alpha; and 9.6 and 540 for I2. Prostaglandin A2, B2, and E1 could not serve as substrate. Although other NADP-linked 15-hydroxyprostaglandin dehydrogenases were reported to contain the NADPH-linked prostaglandin E 9-ketoreductase activity, this enzyme was free of the reductase activity. These results indicate that this dehydrogenase is responsible for the specific inactivation of prostaglandin D2 in the brain.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D006911:Hydroxyprostaglandin Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D009249:NADP; D015230:Prostaglandin D2; D011453:Prostaglandins / Q000378:metabolism*; D011457:Prostaglandins D / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D000596:Amino Acids; D011453:Prostaglandins; D011457:Prostaglandins D; D009249:NADP; D006911:Hydroxyprostaglandin Dehydrogenases; D015230:Prostaglandin D2