猪心2-氧代戊二酸脱氢酶复合体三种组分酶的快速简易分离方法。

Rapid and simple isolation procedure for three component enzymes of pig heart 2-oxoglutarate dehydrogenase complex.

作者信息

Moriyasu M, Koike K, Urata Y, Tsuji A, Koike M

出版信息

J Nutr Sci Vitaminol (Tokyo). 1986 Feb;32(1):33-40. doi: 10.3177/jnsv.32.33.

DOI:10.3177/jnsv.32.33

Abstract

A novel procedure was developed for rapid separation of the three component enzymes of pig heart 2-oxoglutarate dehydrogenase complex by high performance liquid chromatography on a gel filtration column. The complex was dissociated and separated into two fractions of the first dihydrolipoamide succinyltransferase and a second yellow fraction within 1 h by chromatography on a preparative TSK-GEL G4000SW column equilibrated with 0.05 M potassium phosphate buffer (pH 7.0) containing 0.7 M guanidine hydrochloride, 0.05% Triton X-100 and 2 mM dithiothreitol at 10 degrees C. The dihydrolipoamide succinyltransferase fraction was further purified by incubation with 0.5% sodium deoxycholate and subsequent ammonium sulfate fractionation. The other two component enzymes, 2-oxoglutarate dehydrogenase and lipoamide dehydrogenase were separated from the second yellow fraction by chromatography on a calcium phosphate gel-cellulose column. The TSK-GEL column permitted very rapid dissociation and separation of the three component enzymes accompanied by good preservation of their activities and high overall yields.

摘要

开发了一种新方法，通过在凝胶过滤柱上进行高效液相色谱，快速分离猪心2-氧代戊二酸脱氢酶复合体的三种组成酶。在10℃下，用含有0.7M盐酸胍、0.05% Triton X-100和2mM二硫苏糖醇的0.05M磷酸钾缓冲液（pH 7.0）平衡的制备型TSK-GEL G4000SW柱进行色谱分离，1小时内可将该复合体解离并分离成第一部分二氢硫辛酰胺琥珀酰转移酶和第二部分黄色组分。通过与0.5%脱氧胆酸钠孵育并随后进行硫酸铵分级分离，进一步纯化二氢硫辛酰胺琥珀酰转移酶组分。通过在磷酸钙凝胶-纤维素柱上进行色谱分离，从第二部分黄色组分中分离出另外两种组成酶，即2-氧代戊二酸脱氢酶和硫辛酰胺脱氢酶。TSK-GEL柱能够非常快速地解离和分离这三种组成酶，同时很好地保留它们的活性并获得较高的总产率。