Flavonoid glycoside: a new inhibitor of eukaryotic DNA polymerase alpha and a new carrier for inhibitor-affinity chromatography.
Two flavonoid glycosides, kaempferol 3-O-(6"-acetyl)-beta-glucopyranoside (KAG) and quercetin 3-O-(6"-acetyl)-beta-glucopyranoside (QAG), were found to be inhibitors of eukaryotic DNA polymerases from a Japanese vegetable, Petasites japonicus. These compounds inhibited the activities of mammalian replicative DNA polymerases (i.e., pol alpha, delta, and epsilon), but not other pol beta, eta, kappa, and lambda activities. KAG was a stronger inhibitor and more selective to pol alpha than QAG. The IC(50) values of KAG for pol alpha, delta, and epsilon were 41, 164, and 127 microM, respectively. The pol alpha inhibition by KAG was non-competitive with respect to both the DNA template-primer and the dNTP substrate. KAG and QAG did not influence the activities of prokaryotic DNA polymerases or other mammalian DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, human telomerase, human DNA topoisomerase I and II, T7 RNA polymerase, and bovine deoxyribonuclease I. Therefore, we concluded that these flavonoid glycosides are moderate replicative DNA polymerase inhibitors leaning more relatively to pol alpha, and could be used as chromatographic carriers to purify the DNA polymerases rather than cytotoxic agents. We then made a KAG-conjugated column such as the epoxy-activated Sepharose 6B. In the column, pol alpha was selectively adsorbed and eluted.
Mizushina
Yoshiyuki
Y
Laboratory of Food and Nutritional Sciences, Department of Nutritional Science and High Tecnology Research Center, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan. mizushin@nutr.kobegakuin.ac.jp
Ishidoh
Tomomi
T
Kamisuki
Shinji
S
Nakazawa
Satoshi
S
Takemura
Masaharu
M
Sugawara
Fumio
F
Yoshida
Hiromi
H
Sakaguchi
Kengo
K
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism*; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D044949:Kaempferols; D015394:Molecular Structure; D036901:Petasites / Q000737:chemistry; D011485:Protein Binding; D011794:Quercetin / Q000031:analogs & derivatives; D020349:Surface Plasmon Resonance
D004791:Enzyme Inhibitors; D005419:Flavonoids; D006027:Glycosides; D044949:Kaempferols; C006552:kaempferol; D011794:Quercetin; D004256:DNA Polymerase I; D019951:DNA Polymerase beta