title: 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.
abstract: 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-nitrosoguanidine 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 respectively. 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 significant extent. The short half-life of the inactivated alkyltransferase 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.
authors.affiliation: Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey 17033.
authors.forename: A E
authors.initials: AE
authors.lastname: Pegg
authors.forename: L
authors.initials: L
authors.lastname: Wiest
authors.forename: C
authors.initials: C
authors.lastname: Mummert
authors.forename: L
authors.initials: L
authors.lastname: Stine
authors.forename: R C
authors.initials: RC
authors.lastname: Moschel
authors.forename: M E
authors.initials: ME
authors.lastname: Dolan
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000032:analysis; D015153:Blotting, Western; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000031:analogs & derivatives* / Q000633:toxicity; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D014407:Tumor Cells, Cultured / Q000187:drug effects*
chemical_list: D000906:Antibodies; C064976:O(6)-benzylguanine; D008769:Methylnitronitrosoguanidine; D006147:Guanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase