Bovine thymus poly(adenosine diphosphate ribose) polymerase.
About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.
D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008722:Methods; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D009691:Nucleic Acid Denaturation; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D013950:Thymus Gland / Q000201:enzymology*
D006657:Histones; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase