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 chromatography (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 X 10(-2) unit/mg in the initial supernatant to 1272 X 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 degrees C retained 80% of its activity. Michaelis constant for PGE2 was 0.4 microM at pH 7.0 with NAD as a cofactor. These properties of PGDH were compared with the results reported in other literature.
Nagai
K
K
Tsuruta
K
K
Mori
N
N
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000302:isolation & purification*; D008970:Molecular Weight; D009243:NAD; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
D009243:NAD; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase