Uracil-DNA glycosylase in HeLa S3 cells: interconvertibility of 50 and 20 kDa forms and similarity of the nuclear and mitochondrial form of the enzyme.
Uracil-DNA glycosylase activity in HeLa S3 cells was found in nuclei (70%), mitochondria (15%) and cytosol (15%) after fractionation in hypotonic buffers. After fractionation in isotonic buffers the activity in cytosol was increased, apparently as a consequence of leakage from the nuclei. Both in the nuclear and the mitochondrial fraction, a major 50 and a minor 18 kDa form were found after gel filtration in the presence of 0.5 M NaCl. However, after glycerol, gradient sedimentation or gel filtration in the presence of 2 M NaCl or 20% glycerol most of the 50 kDa form dissociated into a 22 kDa form, which was also the smallest catalytically active form found after partial trypsin digestion. The dissociation of the 50 kDa form was reversible. Biochemical properties of the nuclear and mitochondrial forms were very similar. Thus, they had similar apparent Km values, pH optima, heat sensitivities and activation energies, and were stimulated 2-5-fold by 40-60 mM monovalent salt.
D002467:Cell Nucleus / Q000201:enzymology*; D045647:DNA Glycosylases; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D051981:Uracil-DNA Glycosidase
D046911:Macromolecular Substances; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase