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A new method for the assay of poly(adenosine diphosphate ribose) glycohydrolase activity

1975; Elsevier BV; Volume: 66; Issue: 2 Linguagem: Inglês

10.1016/0003-2697(75)90611-9

1096-0309

Luis O. Burzio, Patricio Riquelme, S. S. Koide,

Adenosine and Purinergic Signaling

Poly(adenosine diphosphate ribulose) [poly(ADP-Rib)] glycohydrolase activity was determined by measuring the amount of ADP-Rib hydrolyzed from polymers of ADP-Rib as substrate. In principle, the method consists of incubating oligomers or polymers of [14C]ADP-Rib with testis glycohydrolase. The reaction was stopped by the addition of a suspension of Dowex 1X-2 formate in H2O (1:3, vv) which adsorbed monomers and oligomers of ADP-Rib. The adsorbed [14C]ADP-Rib was selectively extracted from the resin with 6 m formic acid. The amount of [14C]ADP-Rib was estimated by measuring the radioactivity in aliquots of formic acid extract. Oligomers or polymers of ADP-Rib can be utilized as substrates since the reaction rates were the same with either compound. A method to determine phosphodiesterase and glycohydrolase activities was established. These two enzymic activities were distinguished by treating the products of the reactions with alkaline phosphatase and by differential extraction of the adsorbed reaction products on Dowex with 0.5 m and 6 m formic acid.