In vitro hybridization and separation of hybrids of human adenylosuccinate lyase from wild-type and disease-associated mutant enzymes.
In vitro hybridization and separation of hybrids of human adenylosuccinate lyase from wild-type and disease-associated mutant enzymes.
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DOI：
10.1021/bi101734q
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发表时间：
2011-03-01
期刊：
影响因子：
2.9
通讯作者：
Colman RF
中科院分区：
文献类型：
--
作者：
De Zoysa Ariyananda L;Antonopoulos C;Currier J;Colman RF
人腺苷酸琥珀酸裂解酶（ASL）缺乏症是一种遗传性代谢疾病，大多数患者是 ASL 基因突变的复合杂合子。从纯化的野生型和单突变人类 ASL 开始，我们产生了模仿复合杂合子 ASL 的体外杂交体。在本研究中，我们使用了 His-tag WT/non-His-tag WT、His-tag WT/non-His 标签 R396C、His-tag WT/non-His 标签 R396H、His-tag R194C/non-His 标签 R396C 和 His-tag L311V/non-His 标签 R396H 酶对。我们通过使 1 M 氯化胍中的酶对变性并通过去除变性剂使它们复性来产生各种杂交体。根据酶四聚体中存在的组氨酸标签数量，在镍-次氮基-三乙酸-琼脂糖柱上分离杂交体。超速离心分析数据表明，杂种具有大量的异四聚体。对杂种的 Vmax 值的分析表明，大多数亚基行为独立；然而，杂合四聚体保留了弱正协同性，表明不同亚基类型之间存在一定的相互作用。 WT和突变亚基之间的相互作用可能有利于ASL缺陷患者的父母，而一些突变亚基之间的相互作用可能有助于杂合子ASL缺陷患者。
Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild type and single mutant human ASL, we generated in vitro hybrids which mimic compound heterozygote ASL. For this study, we used His-tag WT/non-His-tag WT, His-tag WT/non-His tag R396C, His-tag WT/non-His tag R396H, His-tag R194C/non-His tag R396C, and His-tag L311V/non-His tag R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a Ni-nitrilo-triacetic acid-agarose column based on the number of His-tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the Vmax values of the hybrids indicate that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.