Pronase-Based Assay Method forO⁶-Methylguanine–DNA Methyltransferase

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• 作者：Lim ; In Kyoung ; Kim ; Young Ju ; Kim ; Sangduk ; Paik ; Woon Ki
• 刊名：Analytical Biochemistry
• 出版年：1996
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：May 1, 1996
• 卷：236
• 期：2
• 页码：284-289
• 文件大小：102 K

摘要

A new, simple, and rapid assay method forO⁶-methylguanine–DNA methyltransferase (MGMT) has been developed. When [methyl-³H] DNA radiolabeled withN-[methyl-³H]-N-nitrosourea was incubated together with tissue homogenate, [methyl-³H] group was transferred to the enzyme, formingS-[methyl-³H]cysteine. In contrast to the previous methods which determined the amount of [methyl-³H] group removed from [methyl-³H] DNA, the present method measured the amount of [methyl-³H] transferred to the enzyme. This has been done by hydrolyzing the radiolabeled enzyme with pronase which is a proteolytic enzyme with a broad substrate specificity. On pronase digestion, [methy-³H]-labeled enzyme becomes soluble in trichloroacetic acid. The method is very simple and rapid, and the only expensive equipment required is a scintillation counter which is a relatively routine piece of equipment at present. More than a dozen samples can be processed within 4–5 h without any difficulty. This new method has been employed in the studies on organ distribution of MGMT of rat and mouse.