Catalytic activities of human poly(ADP-ribose) polymerase from normal and mutagenized cells detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
An activity gel procedure is described to identify functional polypeptides of human poly(ADP-ribose) polymerase. Purified or crude enzyme preparations from HeLa cells were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels containing gapped DNA. After renaturation of the peptides in situ, the intact gel was incubated in a poly(ADP-ribose) polymerase reaction mixture containing [32P]NAD. Autoradiograms of the gels consistently exhibited a major activity band at Mr = 116,000-120,000; in many runs, three minor distinct bands at Mr = 125,000, 135,000, and 145,000 were also seen. [32P]NAD appeared to be incorporated into poly(ADP-ribose) since: (i) the activity bands were not detectable when the enzyme-inhibitor 3-aminobenzamide was added to the gel incubation mixture; and (ii) the radioactive polymer, electroeluted from the bands, was completely digested by phosphodiesterase I. Preliminary activity gel analysis of extracts of HeLa cells treated with different DNA-damaging agents revealed that the apparent activity of the Mr = 116,000 form increased by about 10-fold in cells treated with 1 mM dimethyl sulfate and 10-20-fold in cells treated with 10 microM mitomycin C. Only a small increase was obtained in cells treated with 1 mM methyl methanesulfonate, and no change in the activity band pattern was observed after 50 and 100 J/m-2 of UV irradiation.
D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification* / Q000494:pharmacology; D006131:Growth Inhibitors; D006367:HeLa Cells / Q000187:drug effects / Q000201:enzymology / Q000528:radiation effects; D006801:Humans; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D008970:Molecular Weight; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D014466:Ultraviolet Rays
D006023:Glycoproteins; D006131:Growth Inhibitors; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines; D009153:Mutagens; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase