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Metabolism and Enzymology

The Partial Purification and Characterization of Nuclear and Mitochondrial Uracil-DNA Glycosylase Activities from Zea mays Seedlings ¹

Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108

Uracil-DNA glycosylase activities from etiolated Zea mays seedling nuclei and mitochondria were partially purified and characterized. Nuclei and mitochondria were separated using sucrose differential and step gradient centrifugation. Experiments with osmotically shocked organelles indicated that enzyme activity from mitochondria was soluble, whereas nuclear enzyme activity was only partially soluble under the conditions tested. Purification using DEAE-cellulose and Affigel Blue column chromatography yielded distinct elution profiles from both columns for each of the organellar enzyme activities. Final purification was 490- and 850- fold for the nuclear and mitochondrial uracil-DNA glycosylase, respectively. Characterization studies demonstrated significant differences between the nuclear and mitochondrial uracil-DNA glycosylase with respect to K_m, temperature, and pH activity optimum, the effect of salts, and substrate preference. Molecular weight as determined by gel filtration was 18,000 for enzymes from both sources. Both were also sensitive to the sulfhydryl group-blocking agent N-ethylmaleimide. A number of uracil analogs were tested for their ability to inhibit nuclear and mitochondrial uracil-DNA glycosylase activities. 5-Azauracil, uracil, 6-aminouracil, 6-azauracil, 5-aminouracil, and 5-fluorouracil all inhibited both activities to variable degrees.

³ Current address: Building 31, Room 5C19, National Institute on Aging, National Institute of Health, Bethesda, MD 20205.

¹ Supported by research grant DMB-8342894 from the National Science Foundation. This is scientific paper No. 15163 from the Minnesota Agricultural Experiment Station.