Purification of the Death Substrate Poly(ADP-ribose) Polymerase

Thomas Jefferson University, Filadelfia, Pennsylvania, United States
Analytical Biochemistry (Impact Factor: 2.22). 07/1997; 249(1):106-8. DOI: 10.1006/abio.1997.2153
Source: PubMed
Download full-text


Available from: Patrick Duriez, Feb 20, 2015
18 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: There is increasing evidence that the hypersensitive response during plant-pathogen interactions is a form of programmed cell death. In an attempt to understand the biochemical nature of this form of programmed cell death in the cowpea-cowpea rust fungus system, proteolytic activity in extracts of fungus-infected and uninfected cowpea plants was investigated, using exogenously added poly(ADP-ribose) polymerase as a marker. Unlike the proteolytic cleavage pattern of endogenous poly(ADP-ribose) polymerase in apoptotic animal cells, exogenously added poly(ADP-ribose) polymerase in extracts of fungus-infected plants was proteolytically cleaved into fragments of molecular masses 77, 52, 47, and 45 kDa. In vitro and in vivo protease inhibitor experiments revealed the activation of cysteine proteases, and possibly a regulatory role, during the hypersensitive response.
    Experimental Cell Research 01/1999; 245(2):389-99. DOI:10.1006/excr.1998.4256 · 3.25 Impact Factor
  • Source
    Journal of Biological Chemistry 05/2000; 275(20):15504-15511. DOI:10.1074/jbc.275.20.15504 · 4.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: AIP (apoptosis-inducing protein) is a protein purified and cloned from Chub mackerel infected with the larval nematode, Anisakis simplex, which induces apoptosis in various mammalian cells including human tumor cell lines. AIP has shown structural and functional homology to L-amino acid oxidase (LAO) which oxidizes several L-amino acids including L-lysine and AIP-induced apoptosis has been suggested to be mediated by H2O2 generated by LAO activity of AIP. In this study, we confirmed that recombinant AIP generated enough H2O2 in culture medium to induce rapid apoptosis in cells and this apoptosis was clearly inhibited by co-cultivation with antioxidants such as catalase and N-acetyl-cysteine. Surprisingly, however, we found that AIP still could induce H2O2-independent apoptosis more slowly than H2O2-dependent one in HL-60 cells even in the presence of antioxidants. In addition, the HL-60-derived cell line HP100-1, which is a H2O2-resistant variant, underwent apoptosis on treatment with AIP with a similar delayed time course. The latter apoptosis was completely blocked by addition of L-lysine to the culture medium, which is the best substrate of AIP as LAO, indicating that decreased concentration of L-lysine in the culture medium by AIP-treatment induced apoptosis. We also showed that the both apoptosis by AIP were associated with the release of cytochrome c from mitochondria and activation of caspase-9, and overexpressed Bcl-2 could inhibit both of the AIP-induced apoptosis. These results indicate that AIP induces apoptosis in cells by two distinct mechanisms; one rapid and mediated by H2O2, the other delayed and mediated by deprivation of L-lysine, both of which utilize caspase-9/cytochrome c system.
    Cell Death and Differentiation 04/2001; 8(3):298-307. DOI:10.1038/sj.cdd.4400811 · 8.18 Impact Factor
Show more