Differences in environment of FAD between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase shown by active site probe study.
Differences in environment of FAD between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase shown by active site probe study.
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活性位点探针研究显示NAD依赖型和O2依赖型大鼠肝黄嘌呤脱氢酶之间FAD环境的差异。
DOI：
10.1016/s0021-9258(18)71568-8
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
V. Massey
中科院分区：
文献类型：
--
作者：
T. Saito;T. Nishino;V. Massey
通过将天然酶与氯化钙一起孵育来制备大鼠肝脏去黄黄嘌呤脱氢酶。用 FAD 重建后，约 85% 的原始活性得到恢复，所有这些都是 O2 依赖性类型。相反，当二硫苏糖醇处理的去黄素酶与FAD一起温育时，活性的恢复与上述几乎相同，但是大部分恢复的活性是NAD依赖性类型的。预先用或未用二硫苏糖醇处理的去黄酶也用两种人工FAD类似物8-巯基-FAD和6-OH-FAD重构。重构酶和初始去黄酶之间的差异光谱表明，在每种情况下，FAD类似物在二硫苏糖醇处理的酶中以其中性形式结合，而在未经二硫苏糖醇处理的酶中以阴离子形式结合。此外，质子化形式在储存时可以转化为阴离子形式，同时活性从NAD依赖性类型转变为O2依赖性类型。这清楚地表明两种酶蛋白中 FAD 周围的环境不同，这两种酶蛋白通过酶半胱氨酰残基的氧化还原可相互转化。
Rat liver deflavoxanthine dehydrogenase has been prepared by incubating native enzyme with calcium chloride. On reconstitution with FAD, about 85% of the original activity is recovered, all which is the O2-dependent type. In contrast, when dithiothreitoltreated deflavoenzyme is incubated with FAD, the recovery of activity is almost the same as above, but most of the recovered activity is of the NAD-dependent type. Deflavoenzyme with or without previous treatment with dithiothreitol was also reconstituted with two artificial FAD analogues, 8-mercapto-FAD and 6-OH-FAD. The difference spectra between the reconstituted enzymes and the initial deflavoenzyme indicate that, in each case, the FAD analogue is bound in its neutral form in dithiothreitol-treated enzyme, whereas it is bound in the anionic form in enzyme without previous dithiothreitol treatment. Furthermore, the protonated forms can be converted into the anionic forms on storage with a concomitant change of activity from the NAD-dependent to the O2-dependent type. This clearly indicates different environments around FAD in the two types of enzyme protein, which are shown to be interconvertible through oxidation-reduction of enzyme cysteinyl residues.