PURIFICATION OF AN NAD+-DEPENDENT 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE FROM THE HUMAN-PLACENTA

We purified 15-hydroxyprostaglandin dehydrogenase (PGDH) from the human placenta by gel filtration high performance liquid chromatogrpahy (GFHPLC) with spectrophotometric assay for the chromophore of 15-keto PG. Purification steps were as follows; homogenization and centrifugation at 10,000g, for 30 min, ammonium sulfate precipitation, DEAE-cellulose (DE-52), Sephadex G-100 gel filtration, NAD-affinity chromatography (AGNAD type 1), and GFHPLC. The specific activity was increased from 1.5 .times. 10-2 unit/mg in the initial supernatant to 1272 .times. 10-2 after GFHPLC. PGDH showed a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight determined by GFHPLC and electrophoresis was approximately 50,000 daltons. After 4 months of storage, PGDH in a buffer containing 50% glycerol at -20.degree. C retained 80% of its activity. Michaelis constant for PGE2 was 0.4.mu.M at pH 7.0 with NAD as a cofactor. These properties of PGDH were compared with the results reported in other literature.