Purification, characterization and partial peptide microsequencing of progesterone 5 beta-reductase from shoot cultures of Digitalis purpurea
1994
Gartner, D.E. | Keilholz, W. | Seitz, H.U.
Progesterone 5 beta-reductase, which catalyzes the reduction of progesterone to 5 beta-pregnane-3,20-dione, was purified 770-fold to homogeneity from the cytosolic fraction of shoot cultures of Digitalis purpurea. This purification involved DEAE-Sephacel, affinity chromatography (Blue-Sepharose CL-6B and adenosine 2',5'-bisphosphate-Sepharose 4B) and elution from a gel matrix after nondissociating PAGE. The molecular mass determined by SDS/PAGE was 43 kDa and the molecular mass determined by gel-filtration chromatography on calibrated Sephadex G-200 was 280 kDa, thus indicating that the native protein is a polymer consisting of several subunits. The purified enzyme had a Km value of 6 micromolar for NADPH and 34 micromolar for progesterone. The enzyme had a strong substrate specificity for progesterone. The relative rates for other steroids such as testosterone, cortisone and cortisol were much lower. The trypsin digestion of the purified progesterone 54-reductase resulted in 100 peptide fragments. The largest fragment after trypsin digestion and sequence analysis consisted of 13 amino acids.
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