大鼠肝脏中半胱氨酸共轭转氨酶的纯化与特性分析

Purification and characterization of cysteine conjugate transaminases from rat liver.

Tomisawa H, Ichimoto N, Takanohashi Y, Ichihara S, Fukazawa H, Tateishi M

Nippon Roche Research Centre, Kanagawa Prefecture, Japan.

Xenobiotica. 1988 Sep;18(9):1015-28. doi: 10.3109/00498258809042224.

Abstract

Soluble cysteine-conjugate alpha-ketoglutarate transaminase (CAT-I) was purified about 670-fold from rat liver cytosol using s-(p-bromophenyl)-L-cysteine as amino acid substrate. The enzyme preparation of the final step of purification showed a single band in polyacrylamide gel electrophoresis. CAT-I accounted for 64% of the transaminase activity in cytosol. 2. The mol. wt of the enzyme was about 64,000 as determined by gel filtration. Respective Km values for s-(p-bromophenyl)-L-cysteine and alpha-ketoglutaric acid were 1.0 and 1.3 mM in Tris-acetate buffer (pH 7.0). Aminooxyacetic acid, hydroxylamine, and KCN inhibited the enzyme activity. 3. In addition to CAT-I, two isozymes (CAT-IIA and CAT-IIB) were partially purified from rat liver cytosol. In respect of mol. wt, substrate specificity towards cysteine conjugates, and several other properties, CAT-IIA and CAT-IIB were very similar to CAT-I. However, differences were observed for these enzymes in the rate of reverse reaction (formation reaction of cysteine conjugates and alpha-ketoglutaric acid) and substrate specificity towards L-aspartic acid and L-cysteinesulphinic acid.

摘要

以S-（对溴苯基）-L-半胱氨酸作为氨基酸底物，从大鼠肝细胞溶质中纯化出可溶性半胱氨酸共轭α-酮戊二酸转氨酶（CAT-I），纯化倍数约为670倍。纯化最后一步得到的酶制剂在聚丙烯酰胺凝胶电泳中显示为单一条带。CAT-I占细胞溶质中转氨酶活性的64%。2. 通过凝胶过滤测定，该酶的分子量约为64,000。在Tris-乙酸缓冲液（pH 7.0）中，S-（对溴苯基）-L-半胱氨酸和α-酮戊二酸的各自Km值分别为1.0和1.3 mM。氨基氧乙酸、羟胺和氰化钾抑制该酶的活性。3. 除了CAT-I之外，还从大鼠肝细胞溶质中部分纯化出了两种同工酶（CAT-IIA和CAT-IIB）。在分子量、对半胱氨酸共轭物的底物特异性以及其他一些特性方面，CAT-IIA和CAT-IIB与CAT-I非常相似。然而，在逆反应速率（半胱氨酸共轭物和α-酮戊二酸的形成反应）以及对L-天冬氨酸和L-半胱氨酸亚磺酸的底物特异性方面，观察到了这些酶之间的差异。