[Steroid-transforming enzymes from microorganisms. IX. Affinity chromatographic preparation and studies of the apoenzyme from a 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase from Nocardia opaca].
From the flavoenzyme, 4-en-3-oxosteroid: (acceptor)-1-en-oxidoreductase of Nocardia opaca, prosthetic group and apoenzyme were separated quantitatively by means of affinity chromatography in the presence of 2 M (NH4)2 at pH 3.0. Subsequently the apoenzyme was eluted from affinity matrix by 0.01 M phosphate buffer, pH 8.0, whereas under these conditions the intact enzyme could not be eluted. The whole enzyme activity applied could be restored by incubation of the eluted apoenzyme with FAD. The binding strength of the apoenzyme to the immobilized steroid ligand is highly decreased in comparison to the native enzyme and can be interpreted by the action of rest hydrophobicity. That indicates the essential character of FAD for both ligand binding and transformation.
D000645:Ammonium Sulfate; D001051:Apoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D001059:Apoproteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004793:Enzyme Reactivators; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009615:Nocardia / Q000201:enzymology*; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism
D001051:Apoenzymes; D001059:Apoproteins; D004793:Enzyme Reactivators; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase; D000645:Ammonium Sulfate
Steroidumwandelnde Enzyme aus Mikroorganismen. IX. Affinitätschromatographische Präparation und Untersuchung des Apoenzyms einer 4-En-3-oxosteroid: (Akzeptor)-1-en-oxidoreduktase aus Nocardia opaca.