title: Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'- exonuclease activity.
abstract: We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10-13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3'-5'-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.
authors.affiliation: a Department of Science , Systems and Models, Roskilde University , Roskilde , Denmark.
authors.forename: Louise Slot
authors.initials: LS
authors.lastname: Christiansen
authors.affiliation: a Department of Science , Systems and Models, Roskilde University , Roskilde , Denmark.
authors.forename: Gabriella
authors.initials: G
authors.lastname: van Zanten
authors.affiliation: a Department of Science , Systems and Models, Roskilde University , Roskilde , Denmark.
authors.forename: Dvora
authors.initials: D
authors.lastname: Berenstein
authors.affiliation: a Department of Science , Systems and Models, Roskilde University , Roskilde , Denmark.
authors.forename: Marianne
authors.initials: M
authors.lastname: Lauridsen
authors.affiliation: b Department of Biology , University of Copenhagen , Copenhagen , Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Kjærulff
authors.affiliation: b Department of Biology , University of Copenhagen , Copenhagen , Denmark.
authors.forename: Leif
authors.initials: L
authors.lastname: Søndergaard
authors.affiliation: a Department of Science , Systems and Models, Roskilde University , Roskilde , Denmark.
authors.forename: Birgitte
authors.initials: B
authors.lastname: Munch-Petersen
mesh_terms: D000818:Animals; D004256:DNA Polymerase I / Q000737:chemistry; D029721:Drosophila Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004331:Drosophila melanogaster / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D011494:Protein Kinases / Q000737:chemistry*
chemical_list: D029721:Drosophila Proteins; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D004256:DNA Polymerase I
keywords: Enzyme inhibition; enzymology; nucleoside and nucleotide kinases