Degradation of (ADP-ribose)n in permeabilized HeLa cells

Determination of (ADP-ribose)n degradation rates in permeabilized HeLa cells, measured as loss of acid-insoluble radioactivity from permeabilized cells previously incubated with [3H]NAD+, showed bi-phasic kinetics. The majority of label was lost within 20 min at pH 6.0 and 37 degrees C and has a half-life of about 12-15 min. The minor ADP-ribose component was either removed very slowly, or appeared to be stable over an 80 min incubation. The degradation rate of the labile component was directly proportional to the initial amount of ADP-ribose present, and was independent of the experimental conditions used to create various elevated levels. The degradation rates of monomeric and oligo/polymeric ADP-ribose were the same, surprising since different enzymes catalyse the respective reactions. The more stable ADP-ribose component could be more inaccessible to degrading enzymes and/or might represent a different linkage to protein, the cleavage of which is slow.