Affinity chromatography of 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. Use of N,N-dimethylformamide to prevent hydrophobic interactions between the enzyme and the ligand.
1. The 3alpha-hydroxysteroid: NAD+-oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) has been purified by affinity chromatography on Sepharose 4B using glycocholic acid as ligand covalently bound through its carboxyl group to the ethylenediamine spacer. 2. The attachment of the enzyme to the substrate-containing matrix is greatly enhanced by the presence of NAD+ suggesting that this enzyme has a compulsory ordered mechanism where NAD+ binds to the enzyme before the steroid. 3. A NAD+-independent interaction between the enzyme and the ligand was also found. This interaction was mainly hydrophobic and interfered with the NAD+-dependent binding. The NAD+-independent interaction was reduced by N,N-dimethylformamide. 4. By using the affinity column in the presence of 10% N,N-dimethylformamide, highly purified enzyme, as judged from polyacrylamide gel electrophoresis, could be obtained in one step from crude bacterial extracts.
D002846:Chromatography, Affinity; D004126:Dimethylformamide / Q000494:pharmacology*; D005029:Ethylenediamines; D006000:Glycocholic Acid; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D012685:Sepharose
D005029:Ethylenediamines; D009243:NAD; D004126:Dimethylformamide; D012685:Sepharose; D006913:Hydroxysteroid Dehydrogenases; D006000:Glycocholic Acid