Identification of 8-hydroxyguanine glycosylase activity in mammalian tissues using 8-hydroxyguanine specific monoclonal antibody.
Here we report the finding of glycosylase activity in mammalian tissues to remove 8-hydroxyguanine residues from DNA. To detect this activity, a synthetic duplex DNA containing an 8-hydroxyguanine residue was used as the substrate, and before the product was analyzed by HPLC-electrochemical detection (ECD), 8-hydroxyguanine released by the enzymatic action was specifically collected from reactions using immunoaffinity columns of 8-hydroxyguanine specific monoclonal antibody. With the application of immunoaffinity column chromatography, a single peak of 8-hydroxyguanine free from many interfering peaks of unknown origin was clearly demonstrated in HPLC-ECD even with crude tissue extracts used as assay materials. All the tissues of the rats examined showed the activity, suggesting its presence is ubiquitous. The present coupled technique of immunoaffinity column chromatography and HPLC-ECD will be very useful to many future studies of 8-hydroxyguanine glycosylase such as its activity detection, characterization and purification, etc.
Lee
Y S
YS
Department of Pharmacology, Seoul National University College of Medicine, Korea.
Lee
H S
HS
Park
M K
MK
Hwang
E S
ES
Park
E M
EM
Kasai
H
H
Chung
M H
MH
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009699:N-Glycosyl Hydrolases / Q000032:analysis / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D009928:Organ Specificity; D051381:Rats; D017207:Rats, Sprague-Dawley; D013154:Spleen / Q000201:enzymology
D000911:Antibodies, Monoclonal; D009838:Oligodeoxyribonucleotides; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase