Purification of nuclear and mitochondrial uracil-DNA glycosylase from rat liver. Identification of two distinct subcellular forms.
Rat liver uracil-DNA glycosylase has been purified from nuclear extracts over 3000-fold to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a monomeric protein with a polypeptide molecular weight of approximately 35 000. It has a native molecular weight of 33 000 as determined by gel filtration chromatography and a sedimentation coefficient of 2.6 S in glycerol gradients. The nuclear enzyme has an alkaline pH optimum and a pI value of 9.3. Nuclear uracil-DNA glycosylase catalyzes the release of free uracil from both single-stranded and double-stranded DNA with the former being the preferred substrate. The enzyme is unable to recognize dUTP, dUMP, or poly(dA-dT) containing a 3'-terminal uracil residue as a substrate. However, internalization of terminal uracil residues by limited chain elongation produced a substrate for the glycosylase. Another species of uracil-DNA glycosylase has been partially purified from mitochondria. This activity differs from the nuclear enzyme in that it has (i) distinctive chromatographic properties, (ii) a lower native molecular weight of 20 000 as determined by molecular sieving, (iii) a distinct NaCl inhibition profile, and (iv) a longer half-life during thermal denaturation.
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D045647:DNA Glycosylases; D004256:DNA Polymerase I / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D009994:Osmolar Concentration; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase
D007527:Isoenzymes; D012965:Sodium Chloride; D004256:DNA Polymerase I; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase