Quantification of Poly ADP-ribose

Enzymes involved in the degradation of poly(ADP-ribose)-protein. 

Efforts have been made to quantify in vivo levels of poly(ADP-ribose). Hilz and co-workers (77,199) were among the first to develop such assays. Isotopically labeled poly(ADP-ribose) of known specific activity was synthesized in vitro and then purified. This material was then added to a crude extract containing poly(ADP-ribose) and the mixture purified to a new constant specific activity. From this number and the known starting specific activity, the in vivo level of poly(ADP-ribose) was determined (199). 

Hilz and co-workers (77) have utilized another technique to quantify in vivo poly(ADP-ribose). The polymer was isolated and digested with poly(ADP-ribose) glycohydrolase. The 5 -AMP produced as product was detected using an optical assay. 

Several procedures have been developed for the isolation of ADP-rihosylated nuclear protein. In one described by Hayaishi and coworkers (162) ADP-ribosylated nuclear proteins were separated by affinity chromatography in 6 M guanidine- HCl on a dihydroxyhoryl polyacrylamide column (162). A very specific interaction of the borate residue with the cis diol portion of ribose rings of the ADP-ribose permits the selective isolation of ADP-ribosylated proteins. Applying this technique to rat liver nuclear proteins, they observed a distribution of ADP-ribosylation between histone and nonhistone proteins, with histone H2B (67%) and Hi (33%) being preferentially modified. 

In a second procedure, poly(ADP-ribose) was first separated from the bulk of the nucleic acids and proteins by dihydroxyboryl-Sepharose affinity chromatography 147,190). The isolated polymer was treated with snake venom phosphodiesterase and bacterial alkaline phosphatase to yield the nucleoside 2, l -ribosyladenosine from internal residues. This product was then treated with chloroacetaldehyde to produce the fluorescent derivative, l,iSr -ethenoribosyladenosine, which was then separated from other derivatized residues by reversed-phase high performance liquid chromatography picomole amounts were quantified by fluorescence detection. This procedure facilitates the accurate determination of minute quantities of endogenous poly(ADP-ribose) (102, 190). Niedergang et al. (147) have also utilized a fluorimetric assay for determination of the enzymatic digestion products of the polymer, ADP-ribose, or iso-ADP-ribose. 

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