Poly (ADP-Ribose) synthetase. Separation and identification of three proteolytic fragments as the substrate-binding domain, the DNA-binding domain, and the automodification domain.
Poly(ADP-ribose) synthetase of Mr = 120,000 is cleaved by limited proteolysis with alpha-chymotrypsin into two fragments of Mr = 54,000 (54K) and Mr = 66,000 (66K). When the native enzyme is modified with 3-(bromoacetyl)pyridine, both portions of the enzyme are alkylated; however, alkylation of the 54K portions of the enzyme is protected by the addition of the substrate, NAD, or its analog, nicotinamide, suggesting that the substrate-binding site is localized in the 54K fragment. When the enzyme previously automodified with a low concentration of [adenine-U-14C] NAD is digested with alpha-chymotrypsin, the radioactivity is detected exclusively in the 66K fragment. The 66K fragment thus labeled is further cleaved with papain into two fragments of Mr = 46,000 and Mr = 22,000. With these two fragments, the label is detected only in the 22K fragment, but not in the 46K fragment. The 46K fragment binds to a DNA-cellulose column with the same affinity as that of the native enzyme, while the 22K fragment and the 54K fragment have little affinity for the DNA ligand. These results indicate that poly (ADP-ribose) synthetase contains three separable domains, the first possessing the site for binding of the substrate, NAD, the second containing the site for binding of DNA, and the third acting as the site(s) for accepting poly(ADP-ribose).
D000818:Animals; D001665:Binding Sites; D001894:Borohydrides; D002250:Carbon Radioisotopes; D002417:Cattle; D002482:Cellulose / Q000031:analogs & derivatives; D002918:Chymotrypsin; D004247:DNA / Q000031:analogs & derivatives; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D013950:Thymus Gland / Q000201:enzymology*; D014316:Tritium
D001894:Borohydrides; D002250:Carbon Radioisotopes; C033190:DNA-cellulose; D010446:Peptide Fragments; D009243:NAD; D014316:Tritium; D002482:Cellulose; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase; D002918:Chymotrypsin; D010206:Papain