Component analysis and characterization of a nuclear deoxyribonucleotidase.
We have previously reported the identification of a novel activity residing in the nuclear fraction of mammalian cells that selectively binds and hydrolyses deoxyribonucleoside triphosphates. Incubation of this protein with [alpha-32P]dATP leads to the appearance of a retarded band relative to free dATP when the reaction is analysed on non-denaturing polyacrylamide gels. We now show that the retarded species comprises the product of dATP hydrolysis (dADP or dAMP) bound to an as yet unidentified species. We have termed this complex the 'product-nucleotide binding particle' or PNBP*. Through a combination of continuous elution polyacrylamide-gel electrophoresis and gel-filtration chromatography, we demonstrate that the hydrolytic activity (dNTPase) is distinct from the radiolabelled species detected in gel-retardation experiments. T.l.c. confirms that the labelled product does not share RF values associated with a range of mono-, di- and tri-phosphate deoxyribonucleotide standards, and gel-filtration experiments suggest a molecular mass for PNBP* of between 2.5 and 3 kDa. The ability of purified PNBP* to retain its nucleotide ligand after a number of denaturing processes suggests that the ligand is covalently bound. The recovery of dNTPase activity from both gel-filtration and ion-exchange chromatography reveals that the as yet unliganded PNBP* (or a precursor form) is associated with the dNTPase enzyme as part of the active complex, prior to addition of dATP.
Ford
K
K
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Waltho
J
J
Hornby
D
D
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006868:Hydrolysis; D008099:Liver / Q000648:ultrastructure*; D008970:Molecular Weight; D011489:Protein Denaturation; D013552:Swine
D003838:Deoxyadenine Nucleotides