Purification and some properties of 11-hydroxythromboxane B2 dehydrogenase from porcine kidney.

A protein with NAD-dependent 11-hydroxythromboxane B2 dehydrogenase activity was purified to apparent homogeneity from porcine kidney using a relatively simple purification procedure, involving precipitation, anion-exchange chromatography (diethylaminoethyl-cellulose), affinity chromatography (5′-AMP-Sepharose) and gel-filtration chromatography (Protein Pak 125). The dehydrogenase was found to have a molecular mass of 50–55 kDa as determined by comparison with standards on SDS/PAGE. The molecular mass on gel-filtration chromatography was dependent on the ionic strength of the buffer. The apparent Km and Vmax values for thromboxane B2 were also dependent of the ionic strength with a Vmax of 214 nmol min-1 mg-1 using 250 Tris/HC1, pH 8.0, and a corresponding Km of 2.9 mM. The enzyme was NAD dependent and 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, prostaglandin F2α or cholic acid as substrate, and that the enzyme did not catalyse the reverse reaction, conversion of 11-dehydrothromboxane B2 to thromboxane B2. [ABSTRACT FROM AUTHOR]