Pronase-based assay method for O6-methylguanine-DNA methyltransferase.
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.
Lim
K
K
Department of Biochemistry, School of Medicine, Ajou University, Suwon, Korea.
Kim
Y J
YJ
Kim
S
S
Paik
W K
WK
D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis; D004260:DNA Repair; D006868:Hydrolysis; D008745:Methylation; D008780:Methyltransferases / Q000032:analysis*; D051379:Mice; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009928:Organ Specificity; D010455:Peptides / Q000032:analysis*; D011402:Pronase; D051381:Rats; D013997:Time Factors; D014316:Tritium
D010455:Peptides; D014316:Tritium; C008425:S-methylcysteine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011402:Pronase; D003545:Cysteine