Purification to homogeneity of an NAD dependent 11-hydroxythromboxane B2 dehydrogenase from porcine kidney.
A protein with NAD dependent 11-hydroxythromboxane B2 dehydrogenase activity was purified to homogeneity from porcine kidney using a simple purification procedure, involving precipitation, anion exchange chromatography (DE-52), affinity chromatography (5'-AMP-Sepharose) and gel filtration chromatography (Protein Pak 300SW). The dehydrogenase was found to have a molecular mass of 50 kDa and 42 kDa as determined by comparison with standards on SDS/PAGE and gel filtration chromatography respectively. The apparent Km and Vmax values for thromboxane B2 were 220-250 microM and 15-30 nmol min-1 mg-1 respectively. The enzyme was clearly separated from the proteins with 15-hydroxyprostaglandin dehydrogenase activity also present in the kidney. Furthermore it was found that 11-hydroxythromboxane B2 dehydrogenase did not utilize prostaglandin D2 prostaglandin E2 or prostaglandin F2 alpha as substrate, and that the enzyme did not catalyze the reverse reaction, conversion of 11-dehydrothromboxane B2 to thromboxane B2.
Westlund
P
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Department of Reproductive Endocrinology, Karolinska Hospital, Stockholm, Sweden.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D013552:Swine
D009243:NAD; D000429:Alcohol Oxidoreductases; C049239:thromboxane dehydrogenase