Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'- exonuclease activity

Louise Slot Christiansen, Gabriella van Zanten, Dvora Berenstein, Marianne Lauridsen, Søren Kjærulff, Leif Søndergaard, Birgitte Munch-Petersen

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

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
OriginalsprogEngelsk
TidsskriftNucleosides, Nucleotides and Nucleic Acids
Vol/bind35
Udgave nummer10-12
Sider (fra-til)699-766
Antal sider8
ISSN1525-7770
DOI
StatusUdgivet - 1 dec. 2016

Emneord

  • Enzyme inhibition
  • enzymology
  • nucleoside and nucleotide kinases
  • protein kinase
  • 3'-5'-exonuclease

Citer dette

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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.",
keywords = "Enzyme inhibition, enzymology, nucleoside and nucleotide kinases, protein kinase, 3'-5'-exonuclease, inhibition, enzymology, nucleoside and nucleotide kinases, protein kinase, 3'-5'-exonuclease",
author = "{Slot Christiansen}, Louise and Zanten, {Gabriella van} and Dvora Berenstein and Marianne Lauridsen and S{\o}ren Kj{\ae}rulff and Leif S{\o}ndergaard and Birgitte Munch-Petersen",
year = "2016",
month = "12",
day = "1",
doi = "10.1080/15257770.2015.1131295",
language = "English",
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pages = "699--766",
journal = "Nucleosides, Nucleotides and Nucleic Acids",
issn = "1525-7770",
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Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'- exonuclease activity. / Slot Christiansen, Louise; Zanten, Gabriella van; Berenstein, Dvora; Lauridsen, Marianne; Kjærulff, Søren; Søndergaard, Leif; Munch-Petersen, Birgitte.

I: Nucleosides, Nucleotides and Nucleic Acids, Bind 35, Nr. 10-12, 01.12.2016, s. 699-766.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'- exonuclease activity

AU - Slot Christiansen, Louise

AU - Zanten, Gabriella van

AU - Berenstein, Dvora

AU - Lauridsen, Marianne

AU - Kjærulff, Søren

AU - Søndergaard, Leif

AU - Munch-Petersen, Birgitte

PY - 2016/12/1

Y1 - 2016/12/1

N2 - 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.

AB - 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.

KW - Enzyme inhibition

KW - enzymology

KW - nucleoside and nucleotide kinases

KW - protein kinase

KW - 3'-5'-exonuclease

KW - inhibition

KW - enzymology

KW - nucleoside and nucleotide kinases

KW - protein kinase

KW - 3'-5'-exonuclease

U2 - 10.1080/15257770.2015.1131295

DO - 10.1080/15257770.2015.1131295

M3 - Journal article

VL - 35

SP - 699

EP - 766

JO - Nucleosides, Nucleotides and Nucleic Acids

JF - Nucleosides, Nucleotides and Nucleic Acids

SN - 1525-7770

IS - 10-12

ER -