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 first step in the metabolism of prostaglandins which is usually associated with physiological inactivation. A highly purified homogenous enzyme preparation from human placenta was used to determine the molecular mass and lack of quaternary structure of the enzyme. Furthermore we have 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 filtration on FPLC, we could estimate a molecular mass of 28 +/- 1 kDa, indicating 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 28740 +/- 30 Da. (5Z,8E,10E)-12-oxo-5,8,10-heptadecatrienoic acid (oxo-HT) could be identified as the only product obtained from the enzymatic reaction with HHT. Quantification of this metabolite was achieved by gas chromatography/tandem mass spectrometry. The calculated enzyme kinetic constants for the formation of the metabolic product [Km (HHT) = 9.68 microM, Vi = 12.78 mU/micrograms] were in agreement with those determined for NADH formation (Km = 7.65 microM, Vi = 11.79 mU/micrograms). 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 aggregation, dehydrogenation of HHT might represent a biological activation step.
Universitäts-Frauenklinik, Münster, Germany.
W
W
Höhl
B
B
Stahl
R
R
Mundkowski
U
U
Hofmann
C O
CO
Meese
U
U
Kuhlmann
W
W
Schlegel
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
D005231:Fatty Acids, Unsaturated; C008033:12-hydroxy-5,8,10-heptadecatrienoic acid; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase