Study of Poly(ADP-ribosyl)ation in Artemia Embryos at Various Stages of Development
Artemia embryos at different stages of development were analyzed for chromatin associated poly(ADP-ribose) synthetase activity. The enzyme was partially purified and characterized to determine the average polymer length and acceptor proteins. Using (3H)-NAD as substrate, the amount of acid insoluble radio-activity bound to protein during incubation was determined. The reaction was dependent on divalent cations and Mg++, Ca++ and Zn++ showed similar activities. However, Mn++ did not support the reaction. The newly synthesized polymers were stripped from their protein acceptors by alkali treatment and purified on a dihydroxyboronate column. The polymers were hydrolyzed with snake venom phosphodiesterase and alkaline phosphomonoesterase to yield adenosine, ribosyladenosine and diribosyladenosine. These products were analyzed by reversed phase high performance liquid chromotography (HPLC) to determine chain lengths and the degree of branching. The chain lengths obtained were similar for all developmental stages studied and this was found to be less than one unit long, suggesting a high rate of polymer breakdown during the synthesis. Acceptor proteins were determined by SDS-polyacrylamide gel electrophoresis and fluorography of the resulting gel. Our results show that during the first 12 hours of development many proteins serve as acceptors of ADP-ribose, while at 24 and 48 hour stages of development, the main acceptor proteins are histones and the polymerase itself.