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. 1984 Jul;159(1):278–282. doi: 10.1128/jb.159.1.278-282.1984

Purification and characterization of a dUTPase from Acholeplasma laidlawii B-PG9.

M V Williams, J D Pollack
PMCID: PMC215625  PMID: 6145699

Abstract

dUTP was purified 120-fold from extracts of Acholeplasma laidlawii B-PG9 by Blue-Sepharose, Phenyl-Sepharose, hydroxyapatite, and DEAE-Sephacel chromatography techniques. The only substrate for the enzyme was dUTP with an apparent Km of 4.5 microM. The only reaction products were dUMP and PPi. The dUTPase did not exhibit any specific divalent cation requirement, but it was inhibited by EDTA. The enzyme was not inhibited by Pi or p-hydroxymercuribenzoate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be 48,000, and its isoelectric point was 5.3. The enzyme was thermostable at 55 degrees C for 1 h. A. laidlawii dUTPase was distinguishable from KB (human epidermoid carcinoma) dUTPase by differences in electrophoretic migration, isoelectric point, and thermostability. The enzyme is important in preventing dUTP from being incorporated into DNA and may have a significant role in both the synthesis of thymidine- and PPi-dependent phosphorylations.

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Selected References

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