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Pronase-based assay method for O6-methylguanine-DNA methyltransferase.

Lim, K; Kim, YJ; Kim, S; Paik, WK
Analytical biochemistry, 236(2):284-289, 1996
Journal Title
Analytical biochemistry
A new, simple, and rapid assay method for O6-methylguanine-DNA methyltransferase (MGMT) has been developed. When [methyl-3H] DNA radiolabeled with N-[methyl-3H]-N-nitrosourea was incubated together with tissue homogenate, [methyl-3H] group was transferred to the enzyme, forming S-[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, [methyl-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.
MeSH terms
AnimalsCysteine/*analogs & derivatives/analysis*DNA RepairHydrolysisMethylationMethyltransferases/*analysisMiceO(6)-Methylguanine-DNA MethyltransferaseOrgan SpecificityPeptides/*analysisPronaseRatsTime FactorsTritium/diagnostic use
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Journal Papers > School of Medicine / Graduate School of Medicine > Biochemistry & Molecular Biology
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