Novel Antimutagenic Proteins in the Edible Mushroom Agrocybe cylindracea

Yoneda Kasane, Shiozawa Akiko, Kitahara Aiko, Takahashi Eizo, Arimoto Sakae, Okamoto Keinosuke, Negishi Tomoe

doi:10.3123/jemsge.34.9

Many studies show that antigenotoxic ingredients are contained in our daily foods, including in a variety of mushrooms. Identification of antigenotoxic factors is expected to lead to the development of cancer-preventing agents. We previously demonstrated heat-unstable antimutagenic activity in a water-soluble extract of the mushroom Agrocybe cylindracea. In this study we show that antimutagenic components were precipitated in 30-40, 40-50 and 50-60% ammonium sulfate fractions of A. cylindracea extracts. The 30-40% and 40-50% precipitates appeared to contain the same substances and showed strong antimutagenic activity against 2-amino3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and N-nitrosodimethylamine in the Ames test, but not in the Drosophila in vivo DNA repair test. In contrast, the 50-60% precipitate showed antigenotoxic activity against MeIQx in Drosophila but little antigenotoxicity in bacteria. Thus, A. cylindracea contains at least two unique antimutagenic components that can be separated by ammonium sulfate precipitation. We attempted to purify the antimutagenic components contained in each ammonium sulfate fraction. The antimutagenic activity of the 30-50% ammonium sulfate fraction was detected in the flow-through fraction after application to a DEAE-Sepharose column. This fraction exhibited a single band of 27 kDa following electrophoretic analysis on a 15% SDS-polyacrylamide gel (SDS-PAGE). Sequence analysis of eight amino acids at the N-terminal of this protein indicated that this is a novel, previously unreported protein. The antimutagenic component(s) in the 50-60% ammonium sulfate fraction was eluted by 0.5 M NaCl from a DEAE-Sepharose column. As several bands were observed following SDS-PAGE analysis, we could not identify the active components in this fraction. The effects of the ammonium sulfate fraction on the activity of CYP enzymes that activate mutagens metabolically were examined: the 30-40% ammonium sulfate fraction showed strong inhibition of CYP1A, whereas the 50-60% fraction showed slight suppression of CYP1A. In conclusion, a 27-kDa protein from A. cylindracea may suppress mutation due to inhibition of the metabolism of indirect mutagens.<br>

Novel Antimutagenic Proteins in the Edible Mushroom Agrocybe cylindracea

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