title: Flavonoid glycoside: a new inhibitor of eukaryotic DNA polymerase alpha and a new carrier for inhibitor-affinity chromatography.
abstract: 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.
authors.affiliation: 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
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Mizushina
authors.forename: Tomomi
authors.initials: T
authors.lastname: Ishidoh
authors.forename: Shinji
authors.initials: S
authors.lastname: Kamisuki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nakazawa
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takemura
authors.forename: Fumio
authors.initials: F
authors.lastname: Sugawara
authors.forename: Hiromi
authors.initials: H
authors.lastname: Yoshida
authors.forename: Kengo
authors.initials: K
authors.lastname: Sakaguchi
mesh_terms: 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
chemical_list: D004791:Enzyme Inhibitors; D005419:Flavonoids; D006027:Glycosides; D044949:Kaempferols; C006552:kaempferol; D011794:Quercetin; D004256:DNA Polymerase I; D019951:DNA Polymerase beta