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首页> 外文期刊>Biochemistry >In Vitro Hybridization and Separation of Hybrids of Human Adenylosuccinate Lyase from Wild-Type and Disease-Associated Mutant Enzymes
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In Vitro Hybridization and Separation of Hybrids of Human Adenylosuccinate Lyase from Wild-Type and Disease-Associated Mutant Enzymes

机译：人腺苷琥珀酸裂合酶的野生型和疾病相关突变酶的体外杂交和分离。

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摘要

Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which thenmajority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Startingnwith purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimicncompound heterozygote ASL. For this study, we used His-taggedWTon-His-taggedWT, His-taggedWTnon-His-tagged R396C,His-taggedWTon-His-tagged R396H,His-tagged R194Con-His-tagged R396C,nand His-tagged L311Von-His-tagged R396H enzyme pairs. We generated various hybrids by denaturingnpairs of enzymes in 1Mguanidinium chloride and renaturing them by removing the denaturant. The hybridsnwere separated on a nickel-nitrilotriacetic acid-agarose column based on the number of His tags present innthe enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts ofnheterotetramers. Analysis of the Vmax values of the hybrids indicates that most of the subunits behavenindependently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is someninteraction between the different subunit types. The interactions between WT and mutant subunits may benadvantageous to the parents of ASL deficient patients, while the interactions between some mutant subunitsnmay assist heterozygote ASL deficient patients.
机译：人腺苷酸琥珀酸裂合酶（ASL）缺乏症是一种遗传性代谢疾病，其中大多数患者是ASL基因中发生突变的复合杂合子。从纯化的野生型（WT）和单突变人ASL开始，我们生成了模仿化合物杂合子ASL的体外杂种。在这项研究中，我们使用了带His标签的WT /未带His标签的WT，带His标签的WT /非His标签的R396C，带His标签的WT /非His标签的R396H，带His标签的R194C /未带His标签的R396C， n个带有His标签的L311V /非带有His标签的R396H酶对。我们通过使1M氯化胍中的酶对变性并通过去除变性剂使其变性来生成各种杂种。基于四聚酶中存在的His标签的数目，在镍-三氟三乙酸-琼脂糖柱上分离杂种。分析性超速离心数据表明，杂种具有主要量的异四聚体。对杂种的Vmax值的分析表明，大多数亚基表现独立。然而，杂合四聚体保持弱的正协同性，表明不同亚基类型之间存在相互作用。 WT与突变亚基之间的相互作用可能对ASL缺陷患者的父母有益，而某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。

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