睾丸酮假单胞菌3α-羟基类固醇脱氢酶的亲和层析。使用N，N-二甲基甲酰胺防止酶与配体之间的疏水相互作用。

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.

Aukrust L E, Norum K R, Skalhegg B A

Biochim Biophys Acta. 1976 Jun 7;438(1):13-22. doi: 10.1016/0005-2744(76)90219-9.

Abstract

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.

摘要

从睾丸酮假单胞菌（ATCC 11996）中提取的3α-羟基类固醇：NAD⁺氧化还原酶（EC 1.1.1.50）已通过在琼脂糖4B上进行亲和层析进行纯化，使用甘氨胆酸作为配体，通过其羧基与乙二胺间隔基共价结合。2. NAD⁺的存在极大地增强了酶与含底物基质的结合，这表明该酶具有强制有序机制，其中NAD⁺在类固醇之前与酶结合。3. 还发现了酶与配体之间的不依赖NAD⁺的相互作用。这种相互作用主要是疏水性的，并干扰了依赖NAD⁺的结合。N,N-二甲基甲酰胺可减少不依赖NAD⁺的相互作用。4. 通过在10% N,N-二甲基甲酰胺存在下使用亲和柱，从粗细菌提取物中一步即可获得经聚丙烯酰胺凝胶电泳判断为高度纯化的酶。