title: Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.
abstract: Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.
authors.affiliation: Bioenergetics Research Centre, CONICET, School of Medicine, University of Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina.
authors.forename: Dolores
authors.initials: D
authors.lastname: Podestá
authors.forename: María I
authors.initials: MI
authors.lastname: García-Herreros
authors.forename: Joaquín J B
authors.initials: JJ
authors.lastname: Cannata
authors.forename: Andrés O M
authors.initials: AO
authors.lastname: Stoppani
authors.forename: Silvia H
authors.initials: SH
authors.lastname: Fernández Villamil
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange; D016831:Crithidia fasciculata / Q000201:enzymology*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D011065:Poly(ADP-ribose) Polymerases; D004264:DNA Topoisomerases, Type I