Enzymatic Properties of Pierisin-1 and Its N-Terminal Domain, a Guanine-Specific ADP-Ribosyltransferase from the Cabbage Butterfly

• Watanabe Masahiko

  Cancer Prevention Basic Research Project, National Cancer Center Research Institute

• Enomoto Shigeki

  Cancer Prevention Basic Research Project, National Cancer Center Research Institute

• Takamura-Enya Takeji

  Cancer Prevention Basic Research Project, National Cancer Center Research Institute

• Nakano Tsuyoshi

  Cancer Prevention Basic Research Project, National Cancer Center Research Institute

• Koyama Kotaro

  Cancer Prevention Basic Research Project, National Cancer Center Research Institute

• Sugimura Takashi

  Cancer Prevention Basic Research Project, National Cancer Center Research Institute

• Wakabayashi Keiji

  Cancer Prevention Basic Research Project, National Cancer Center Research Institute

抄録

The cabbage butterfly, Pieris rapae, produces an ADP-ribosylating cytotoxic protein, pierisin-1. Unlike other ADP-ribosylating toxins, the acceptor site for ADP-ribosylation by pierisin-1 is the N-2 position of guanine bases in DNA. The present study was designed to characterize this novel guanine-specific ADP-ribosyltransferase, pierisin-1. The N-terminal polypeptide from Met-1 to Arg-233, but not the C-terminal Ser-234-Met-850 polypeptide, was found to exhibit guanine ADP-ribosyltransferase activity. Trypsin-treated pierisin-1, which is considered to be a “nicked” full-length form composed of associated N- and C-terminal fragments, also demonstrated such activity. Optimum conditions for the N-terminal polypeptide of pierisin-1 were pH 8-10, 37-40°C, in the presence of 100-200mM NaCl or KCl. Other metal ions such as Ca²⁺ or Mg²⁺ were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a K_M value for NAD of 0.17mM and k_cat of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (k_cat=25 per second). When the conditions were changed to pH 5-7 or 10-20°C, some activity (6-55% or 5-20%, respectively, of that under optimal conditions) of the N-terminal polypeptide was still evident; however, almost all of the trypsin-treated enzyme activity disappeared. This implies the inhibition of the N-terminal enzyme domain by the associated C-terminal fragment. Long-term reactions indicated that a single molecule of pierisin-1 has the capacity to generate more than 10⁶ ADP-ribosylated DNA adducts, which could cause the death of a mammalian cell.

収録刊行物

• The Journal of Biochemistry

  The Journal of Biochemistry 135 (4), 471-477, 2004

  社団法人 日本生化学会

キーワード

• cabbage butterfly
• DNA adduct
• guanine ADP-ribosyltransferase
• pierisin-1
• toxin

詳細情報

• CRID: 1390282679943112320
• NII論文ID: 130003534685
• NII論文ID: 10016199301
• NII書誌ID: AA00694073
• DOI: 10.1093/jb/mvh062
• COI: 1:CAS:528:DC%2BD2cXltFCgt7c%3D
• ISSN: 17562651
• ISSN: 0021924X
• NDL書誌ID: 6931130
• PubMed: 15115771
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I6931130
• Web Site: http://academic.oup.com/jb/article-pdf/135/4/471/2904264/mvh062.pdf
• Web Site: https://search.jamas.or.jp/link/ui/2005013568
• 本文言語コード: en
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
• 抄録ライセンスフラグ: 使用不可