Affordable Access

Publisher Website

Pronase-Based Assay Method forO6-Methylguanine–DNA Methyltransferase

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
236
Issue
2
Identifiers
DOI: 10.1006/abio.1996.0168
Disciplines
  • Biology

Abstract

Abstract A new, simple, and rapid assay method for O 6-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, [ 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.

There are no comments yet on this publication. Be the first to share your thoughts.