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J. Biochem, 2004, Vol. 135, No. 4 471-477
© 2004 The Japanese Biochemical Society

BIOCHEMISTRY

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

Masahiko Watanabe*, Shigeki Enomoto, Takeji Takamura-Enya, Tsuyoshi Nakano, Kotaro Koyama, Takashi Sugimura and Keiji Wakabayashi

Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045

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 100–200 mM NaCl or KCl. Other metal ions such as Ca2+ or Mg2+ were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a KM value for NAD of 0.17 mM and kcat of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (kcat = 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.

* To whom correspondence should be addressed. Tel: +81-3-3542-2511, Fax: +81-3-3543-9305, E-mail: mwatanab{at}gan2.ncc.go.jp


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