Use of antibodies to human O6-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O6-benzylguanine or N-methyl-n'-nitro-N-nitrosoguanidine

Anthony E. Pegg, Laurie Wiest, Christine Mummert, Linda Stine, Robert C. Moschel, M. Eileen Dolan

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Antisera raised in rabbits to three peptides corresponding to amino acid sequences found in human O6 -alkylguanine-DNA alkyltransferase were used to study the fate of the alkyltransferase protein in human colon tumor cells after exposure to N-methyl-N'-nitro-N-ntrosoguanidine or to O6 -benzylguanidine. Under these conditions, the alkyltransferase protein becomes inactivated, presumably by the conversion of its cysteine acceptor site to S-methylcysteine or S-benzylcysteine rrspectively. It was found that the protein was rapidly degraded after such inactivation both in intact cells and in cell-free extracts. It is probable that a conformational change in the protein is brought about by conversion of the alkyltransferase to the Inactive form by alkylation of the cysteine acceptor site. This change may render the protein very sensitive to proteolytic degradation. The rapid degradation of the inactive form of the protein may serve as a signal for its resynthesis but in the short term ensures that its reactivation by regeneration of the cysteine acceptor site is unlikely to occur to any sigificant extent. The short halflife of the inactivated alkyltranferase protein makes it probable that measurement of the content of the alkyltransferase protein by immunohistochemistry, which is likely to measure the sum of the active and inactivated forms of the protein, will nevertheless yield an accurate estimation of the cellular capacity to repair O6-methylguanine provided that procedures with sufficient specificity and affinity can be developed.

Original languageEnglish (US)
Pages (from-to)1679-1683
Number of pages5
Issue number9
StatePublished - Sep 1991

All Science Journal Classification (ASJC) codes

  • Cancer Research


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