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 hydro-xylamine. 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-ribo-sylated. 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.