Purification, characterization and partial peptide microsequencing of progesterone 5 beta-reductase from shoot cultures of Digitalis purpurea.
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 non-dissociating 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 microM for NADPH and 34 microM 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 5 beta-reductase resulted in 100 peptide fragments. The largest fragment after trypsin digestion and sequence analysis consisted of 13 amino acids.
Gärtner
D E
DE
Botanisches Institut der Universität, Tübingen, Germany.
Keilholz
W
W
Seitz
H U
HU
D000595:Amino Acid Sequence; D002298:Cardenolides / Q000378:metabolism; D004070:Digitalis / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010946:Plants, Medicinal; D010947:Plants, Toxic; D013256:Steroids; D013379:Substrate Specificity
D002298:Cardenolides; D010446:Peptide Fragments; D013256:Steroids; D009249:NADP; D010088:Oxidoreductases; C090493:progesterone 5 beta-reductase