A method was developed for the isolation of protein ADP-ribosylated in vivo, using mouse L1210 cells. The method exploited the differences in buoyant density of RNA/DNA and protein on caesium chloride density gradient centrifugation. In order that the method may be generally applicable, it was designed to be both non-selective and to ensure full recovery of ADP-ribosylated protein. Approximately 13% of the [3H] adenosine-labelled, acid-insoluble material banded at the top of the gradient and was assumed to be ADP-ribosylated protein. The isolated ADP-ribosylated protein was characterized, using enzymic and chemical analyses, with respect to chain length, ADPR-protein linkage, and number and type of proteins modified. The presence of PR-AMP after snake venom phosphodiesterase digestion confirmed the existence of poly(ADP-ribosyl)ated protein in vivo. The characterization showed that the majority (approx. 85%) of material at the top of the gradient represented ADP-ribosylated protein. The average chain length of ADPR bound to protein was estimated as 1.3 ADPR units. 87% of material on top of the gradient was sensitive to base and 21% was sensitive to hydroxyl amine. Of the material which remained acid-insoluble after treatment with base, approximately 55% represented base-resistant ADP-ribosylated protein. This suggests that there may be at least three types of linkage between ADPR and protein in vivo. Analysis of proteins by SDS gel electrophoresis revealed that a number of species were ADP-ribosylated. To characterize the in vivo system further, the effects of 3-aminobenzamide and thymidine, which are known to inhibit poly(ADPR) synthetase in vitro, were studied. In contrast to results obtained in vitro, only partial inhibition of ADP-ribosylation was observed. Furthermore, the extent of inhibition appeared to decrease with time, being 65% at 6 hours but only 40% at 24 hours. The effect of the alkylating agent dimethyl sulphate on ADP-ribosylation in vivo was also studied. Addition of 105 muM DMS resulted in a rapid but transitory decrease in NAD+ levels and a complementary increase in ADP-ribosylation of protein. This finding supports the hypothesis that ADP-ribosylation of proteins in vivo may be involved in DNA repair.
|Date of Award||1981|