Properties of purified calf thymus poly(adenosine diphosphate ribose) polymerase. Comparison of the DNA-independent and the DNA-dependent enzyme.
The physicochemical properties of the purified calf thymus poly(ADP-ribose) polymerase were investigated. The enzyme purified to homogeneity was shown to contain about 10% DNA on a weight basis and its activity to be DNA independent. After removing this fragment of DNA, called the sDNA fraction, the enzyme becomes DNA dependent. The activity of this enzyme preparation was entirely dependent on, and completely restored by, added calf thymus DNA or sDNA. However, the calf thymus DNA concentration needed was a hundred times higher than that of sDNA. The properties of the two enzyme preparations, DNA independent and DNA dependent, were essentially the same. They both reacted against the specific antibody obtained with the DNA-independent poly(ADP-ribose) polymerase. The pH optimum was around 8; the activity was stimulated by Mg2+, Mn2+ and Ca2+, and inhibited by high ionic strength, p-chloromercuribenzoate, ADP-ribose, AMP and polylysine. Nicotinamide, thymidine and NADP were shown to be competitive inhibitors. The enzymatic activity was stimulated by histone H1 when the ratio of DNA to histone H1 was 2. Histones H2A, H2B, H3 and H4 had little effect on the DNA-independent enzyme activity, but were strongly inhibitory for the DNA-dependent enzyme. This inhibitory effect could be reversed by allowing the DNA-dependent enzyme to react with the sDNA fraction before adding histone subfractions. The apparent Km for NAD of the DNA-dependent poly(ADP-ribose) polymerase was shown to vary with the DNA concentration. It was minimum when the amount of sDNA was 10% of that of the enzyme. The ratio of the apparent Km for sDNA to the enzyme concentration was constant at any enzyme concentration. The minimum estimation of the number of base pairs of sDNA required for maximal activation of one enzyme molecule was 16. For calf thymus DNA, this estimation was of 640. These results suggest that the activation of the enzyme needs the formation of some complex between the protein and a specific part of the DNA. This complex was preserved in the DNA-independent enzyme preparation.
D000818:Animals; D002417:Cattle; D002845:Chromatography; D004247:DNA / Q000378:metabolism*; D006657:Histones / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013696:Temperature; D013950:Thymus Gland / Q000201:enzymology*
D006657:Histones; D009711:Nucleotides; D009243:NAD; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase