Xenopus mono(adenosine diphosphate ribosyl) transferase: purification, assay, and properties.

Various tissues of Xenopus laevis possess mono(ADP-ribosyl) transferase activity. The Xenopus liver enzyme was purified approximately 40,000-fold by sequential chromatography on phenyl Sepharose, DEAE-Sephadex, NAD+-Agarose, and Con A-Sepharose. The enzyme is relatively heat-stable, has no cation requirements, transfers monomers of ADP-ribose from NAD+ to arginine-rich histones H3 and H4, and is not dependent upon DNA for activity. Its Mr was estimated to be 30 kilodaltons (KD). The amino acid acceptor of the ADP-ribose moiety is probably arginine, and the bond formed is resistant to the hydrolytic action of neutral hydroxylamine. The transferase activity was neither inhibited nor precipitated by antisera directed against the A fragment of cholera or diphtheria toxins. Theophylline, thymidine, nicotinamide, and some of its analogues blocked the enzymatic activity, while nicotinic acid was ineffective. When a DNA-free extract of HeLa cells was incubated with the transferase system, several proteins were found to be ADP-ribosylated. A band at position 90 KD showed high specific radioactivity. A zymographic method for assaying transferase activity in crude tissue extracts was developed. In the initial step, the extracts were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Using this method, a histone-dependent mono(ADP-ribosyl) transferase corresponding to an Mr of about 30 KD was demonstrated in every tissue examined. Treatment with reducing agents practically abolished transferase activity in contrast to the enhancement of choleragen A fragment activity. A variant of the transferase at about 80 KD was detected. It differed from the 30 KD enzyme in that histones inhibited its activity while agmatine had no influence.