Purification and characterization of 3-methyladenine-DNA glycosylase from calf thymus.
The 3-methyladenine-DNA glycosylase from calf thymus has been purified and characterized. Two species of Mr = 42,000 and 27,000 +/- 5% and Stokes radius of 27.5 and 22.4 A, respectively, were found. Only the lower molecular weight species were present in the nucleus; it was bound to chromatin and could be dissociated in the presence of 0.25 M KCl. The enzymatic properties of the two species appeared to be identical. Both enzyme species released 3-methyladenine, 7-methylguanine, and 3-methylguanine, listed in the order of decreasing activity. The chromatin-associated enzyme was purified to apparent homogeneity and found to be a basic protein having a pI greater than 9. It was completely inhibited by p-hydroxymercuribenzoate, but this inhibition could be fully reversed by addition of excess 2-mercaptoethanol. Kinetic studies, heat inactivation, and inhibition experiments demonstrated that the 3-methyladenine and 7-methylguanine releasing activities were located on the same protein molecule. The enzymes showed no activity on methylated single-stranded DNA. No product inhibition was observed for any of the enzyme species, and the enzyme activity was optimal when the incubation was performed in the presence of 50 mM NaCl or KCl at pH values between 8 and 9.
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002843:Chromatin / Q000201:enzymology; D045647:DNA Glycosylases; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013950:Thymus Gland / Q000201:enzymology
D002843:Chromatin; D013438:Sulfhydryl Compounds; C044502:3-methylguanine; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D000225:Adenine