Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase.
Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.
Mendoza-Alvarez
H
H
Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth 76107-2699, USA.
Alvarez-Gonzalez
R
R
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002843:Chromatin / Q000737:chemistry; D002918:Chymotrypsin / Q000378:metabolism; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D002843:Chromatin; D009243:NAD; D000246:Adenosine Diphosphate Ribose; C057676:3'-deoxynicotinamide adenine dinucleotide; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; D002918:Chymotrypsin; D010206:Papain