11 Pages

Poly(ADP-ribose): Structure, Quantification, and Biological Significance

Nakahara Memorial Lecture
ByMasanao Miwa, Mieko Kato, Hisae Iijima, Yuko Tanaka, Tomoko Kondo, Hisae Kawamitsu, Masaaki Terada, Takashi Sugimura

We have been working on poly(ADP-Rib) since our discovery of it. This lecture will review recent findings on this polymer obtained in our laboratory. Poly (ADP-Rib) has long been thought to be a linear homopolymer attached to nuclear protein. The ribose-ribose bond of poly(ADP-Rib) was shown to be an α(1”→02’) ribosidic bond. There are two molecular forms of poly(ADP-Rib), namely low molecular weight (L) and high molecular weight (H) poly(ADR-Rib) molecules. The L and H fractions of poly(ADP-Rib) are separable by gel filtration, sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. Physicochemically the L and H forms differ in their molecular size, solubility in high salt, rate of hydrolysis by venom phosphodiesterase and circular dichroism (CD) spectrum. However, the two forms have a similar ultraviolet (UV) absorption spectrum, both show a similar hyperchromicity on heating to 98°C or hydrolysis with snake venom phosphodiesterase and give similar results on ordinary chain length determination. The great discrepancy in size observed by physicochemical methods and by the ordinary chain length method indicates the presence of a branching structure in poly(ADP-Rib). The branching structure was proved by demonstrating the presence of a unique compound, 2′[1”-ribosy1-2″(1′″-ribosyl)]adenosine-5’,5″,5′″-tris(phosphate) and by electron microscopy. The amount of poly(ADP-Rib) was determined by our new method, consisting of tritium labeling and high performance liquid chromatography. The merit of this method is that it can determine the recovery of poly(ADP-Rib) exactly and that its sensitivity is high. The amount of poly(ADP-Rib) changed dramatically when human promyelocytic leukemia cells were induced to differentiate by dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate. The use of inhibitors of poly (ADP-Rib) polymerase in combination with bleomycin, a DNA damaging antitumor drug, potentiated the antitumor activity of belomycin against Ehrlich ascites carcinoma cells in vivo.