title: Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver.
abstract: Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.
authors.affiliation: Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
authors.forename: J D
authors.initials: JD
authors.lastname: Domena
authors.forename: R T
authors.initials: RT
authors.lastname: Timmer
authors.forename: S A
authors.initials: SA
authors.lastname: Dicharry
authors.forename: D W
authors.initials: DW
authors.lastname: Mosbaugh
mesh_terms: D000818:Animals; D004247:DNA; D045647:DNA Glycosylases; D004260:DNA Repair; D066298:In Vitro Techniques; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014498:Uracil / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase
chemical_list: D014498:Uracil; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase