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Poly(adenosine diphosphate ribose) glycohydrolase, which has thus far only been found in mammalian tissues, was found for the first time in the primitive eukaryotic slime mold Physarum polycephalum. The hydrolytic product of poly(adenosine diphosphate ribose) with this enzyme was identified as adenosine diphosphate ribose by paper and thin-layer chromatography. It is likely that the enzyme caused exoglycosidic hydrolysis. The optimal pH of this enzyme was 6.0, and the Km value was 4.3 μ m , as adenosine diphosphate ribose residues of polymer. Adenosine diphosphate ribose, ADP and ATP at a concentration of 0.1m m strongly inhibited the enzyme activity. 3′,5′-Cyclic AMP was inhibitory at a concentration of 1m m . The molecular weight of this enzyme was estimated to be 57,000.

ADP-ribosyltransferase activity associated with chromatin is two- to tenfold higher in simian virus 40 (SV40)-transformed cells than in untransformed cells. When confluent transformed cells were subcultured, their specific enzyme activity first decreased two- to fourfold and the rapidly increased during the logarithmic phase of growth. This increase ceased or slowed down when the cells entered the stationary phase. In contrast, the activity in the untransformed cells remained low throughout the growth cycle. In SV40tsA-transformed cells (ts = temperature sensitive), this density-dependent increase in the enzyme activity was observed when the cells were cultivated at the permissive temperature, whereas the activity remained low at the restrictive temperature. The enzyme activity did not increase during induction of cellular DNA synthesis in quiescent cells either by addition of fresh medium or by infection with SV40. The chromatin-associated enzyme activity extracted with 1 m NaCl was eluted together with almost all the DNA-binding proteins from a phosphocellulose column with 0.6 m NaCl. The enzyme activity in this fraction from transformed cells, measured with or without added DNA and histones, was higher than that in a similar fraction from untransformed cells, reflecting the difference in the original activities present in the nuclei of these cells. The chain lengths of poly(ADP-ribose) formed by chromatin from SV40-transformed and untransformed cells were not significantly different. These results suggest that the number of initiation sites for ADP-ribosylation is increased in the chromatin of SV40-transformed cells compared to that of untransformed cells.

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.

Poly(ADP-ribose) glycohydrolase, which was first identified in calf thymus and rat liver, was found to be present in all normal rat tissues and tumors. Of the tissues examined, the testis had the highest activity. The major pathway of degradation of poly(ADP-ribose) in all tissues was through poly(ADP-ribose) glycohydrolase. Phosphodiesterase, which was first found in rat liver, was only of minor importance in hydrolysis of poly(ADP-ribose). These findings suggest the importance of poly(ADP-ribose) glycohydrolase both in normal and tumor tissues for the rapid turnover of poly(ADP-ribose).