通过薄层色谱法对双功能5-氨基咪唑-4-甲酰胺核苷酸转甲酰基酶-IMP环水解酶进行放射测定。
Radioassay of bifunctional 5-aminoimidazole-4-carboxamide ribotide transformylase-IMP cyclohydrolase by thin-layer chromatography.
作者信息
Szabados E, Christopherson R I
机构信息
Department of Biochemistry, University of Sydney, New South Wales, Australia.
出版信息
Anal Biochem. 1994 Sep;221(2):401-4. doi: 10.1006/abio.1994.1433.
A radioassay has been developed for the bifunctional enzyme, AICAR transformylase-IMP cyclohydrolase, which catalyzes reactions 9 and 10 of the de novo pathway for biosynthesis of purine nucleotides (AICAR-->FAICAR-->IMP). 3H-labeled AICAR or FAICAR is converted enzymically to product(s) which are separated by one-dimensional thin-layer chromatography prior to quantification by scintillation counting. Using this sensitive radioassay, a dissociation constant of IMP cyclohydrolase for FAICAR of 0.87 microM has been determined and AICAR, FAICAR, and IMP can be quantified in assay mixtures for AICAR transformylase-IMP cyclohydrolase. The ratio of specific enzymic activities for AICAR transformylase:IMP cyclohydrolase is 1:44.
已开发出一种用于双功能酶——氨基咪唑甲酰胺核糖核苷酸转甲酰酶-肌苷酸环水解酶的放射分析方法，该酶催化嘌呤核苷酸从头合成途径中的反应9和10（氨基咪唑甲酰胺核糖核苷酸→5'-甲酰氨基咪唑-4-甲酰胺核糖核苷酸→肌苷酸）。用³H标记的氨基咪唑甲酰胺核糖核苷酸或5'-甲酰氨基咪唑-4-甲酰胺核糖核苷酸通过酶促反应转化为产物，在通过闪烁计数进行定量之前，先用一维薄层色谱法将产物分离。使用这种灵敏的放射分析方法，已测定出肌苷酸环水解酶对5'-甲酰氨基咪唑-4-甲酰胺核糖核苷酸的解离常数为0.87微摩尔，并且在氨基咪唑甲酰胺核糖核苷酸转甲酰酶-肌苷酸环水解酶的分析混合物中可以对氨基咪唑甲酰胺核糖核苷酸、5'-甲酰氨基咪唑-4-甲酰胺核糖核苷酸和肌苷酸进行定量。氨基咪唑甲酰胺核糖核苷酸转甲酰酶与肌苷酸环水解酶的比酶活性之比为1:44。
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