In Vitro Hybridization and Separation of Hybrids of Human Adenylosuccinate Lyase from Wild-Type and Disease-Associated Mutant Enzymes

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• 作者：Lushanti De Zoysa Ariyananda ; Christina Antonopoulos ; Jenna Currier ; Roberta F. Colman
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：March 1, 2011
• 年：2011
• 卷：50
• 期：8
• 页码：1336-1346
• 全文大小：978K
• 年卷期：v.50,no.8(March 1, 2011)
• ISSN：1520-4995

文摘

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 (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged 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 nickel鈭抧itrilotriacetic acid鈭抋garose 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 V_max values of the hybrids indicates 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.