MASS DETERMINATION OF 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE FROM HUMAN PLACENTA AND KINETIC-STUDIES WITH (5Z, 8E, 10E, 12S)-12-HYDROXY-5,8,10-HEPTADECATRIENOIC ACID AS SUBSTRATE

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the fir st step in the metabolism of prostaglandins which is usually associate d with physiological inactivation. A highly purified homogenous enzyme preparation from human placenta was used to determine the molecular m ass and lack of quaternary structure of the enzyme. Furthermore we hav e examined enzyme kinetics of the purified enzyme with 5Z,8E,10E,12S)- 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) an equimolar coproduct of thromboxane biosynthesis. Using gel electrophoresis and gel filtrat ion on FPLC, we could estimate a molecular mass of 28 +/- 1 kDa, indic ating that the enzyme consists of one single protein chain. The exact molecular mass of the monomer was calculated by matrix-assisted laser desorption/ionization mass spectrometry to 28 740 +/- 30 Da. (5Z,8E,10 E)-12-oxo-5,8,10-heptadecatrienoic acid (oxo-HT) could be identified a s the only product obtained from the enzymatic reaction with HHT. Quan tification of this metabolite was achieved by gas chromatography/tande m mass spectrometry. The calculated enzyme kinetic constants for the f ormation of the metabolic product [K(m) (HHT) = 9.68 muM, V(i) = 12.78 mU/mug] were in agreement with those determined for NADH formation (K (m) = 7.65 muM, V(i) = 11.79 mU/mug). This demonstrates that HHT shows high affinity to the enzyme which is comparable to prostaglandin E2 ( PGE2). As the product oxo-HT is a potent inhibitor of platelet aggrega tion, dehydrogenation of HHT might represent a biological activation s tep.