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Hydroxylapatite column chromatography was used for purification of poly (ADP-Rib). With an increasing concentration gradient of phosphate buffer (pH 6.8) RNA, DNA, and poly (ADP-Rib) with a chain of 17–18 ADP-ribose units were eluted successively. RNase and DNase digestion of a crude extract containing RNA, DNA, and poly(ADP-Rib) before chromatography resulted in a better separation of poly (ADP-Rib). Poly (ADP-Rib) with a shorter chain length were eluted with more dilute phosphate buffer and a linear relationship was obtained between the chain length of the material eluted and the phosphate concentration of the elution buffer.

The enzyme catalyzing the synthesis of poly (adenosine diphosphate-ribose) with an average of eight repetitions of ADP-ribose was purified 10-fold from rat liver nuclei in 15% yield. The enzyme required DNA, histone, MgCl2, and dithiothreitol for activity. DNA could not be replaced by polyanions such as poly (U), poly (A), poly (C), RNA, polyvinyl sulfate, methyl dextran sulfate, or heparin. The enzyme was as active on native DNA as on heat-denatured DNA and on poly [d (A-T)], but less active on poly(dG)·poly(dC) and on acid-soluble oligodeoxyribonucleotide. Whole histones of calf thymus or of rat liver, lysine-rich histone of calf thymus, and arginine-rich histone were similarly effective in stimulating the reaction. Casein, bovine serum albumin, cytochrome c, and spermidine did not replace lysine-rich histone. CaCl2 or MnCl2 was as effective for the reaction as MgCl2. Dithiothreitol could be replaced by 2-mercaptoethanol and by glutathione. Polyanions, such as RNA, poly(U), poly(C), poly(A), and polyvinyl sulfate inhibited the enzyme activity. The molecular weight of the enzyme was determined to be 78,000 by sucrose density gradient centrifugation.

Summary Specific antibody against poly(ADP-Rib) was produced in a rabbit by injecting poly(ADP-Rib) mixed with methylated bovine serum albumin. Under standardized conditions, 1 mg of purified anti-poly(ADP-Rib) antibody combined with 400 pmoles (4 μg) of poly(ADP-Rib) and was retained on a millipore filter. The binding of [ 14 C]poly-(ADP-Rib) was not inhibited by poly(A) or other related nucleotides.

Poly(ADP-Rib) was hydrolyzed by a calf thymus nuclear preparation. The reaction products were ADP-Rib and oligo(ADP-Rib). This reaction is a novel mechanism for degradation of poly(ADP-Rib).