Covalent modification of proteins by ADP-ribosylation
The fields of nuclear and extra-nuclear ADP- ribosylation reactions have been reviewed with a view to clarifying much of the recently published but often conflicting literature. The major reactions reportedly carried out by the nuclear ADPRT have been summarised in the form of two simple diagrams. The reviewed data are consistent with the hypothesis that monomeric and polymeric ADP-ribosylation are unified and interdependent reactions probably carried out by a combination of poly(ADP-ribose) glycohydrolase and a single species of ADP-ribosyl transferase. Polyoma-virus transformed BHK fibroblasts have a greater ADPRT activity than their non-transformed counterparts. The greater rate of polymer synthesis in the transformed cell line is not a consequence of decreased poly(ADP-ribose) degradation. In both cell lines the gross rates of ADP-ribose turnover are similar and are slow relative to other reports. The permeabilized cell technique has been used in conjunction with newly modified procedures for electrophoretically resolving proteins solubilized from BHK and PyY-BHK fibroblasts in order to determine the consequences for protein ADP-ribosylation of the greater enzyme activity in the transformed cell line. Although a number of differences in terms of ADPRT activity under various circumstances have been detected, the increased enzyme activity in the transformed cell line does not appear to result in reproducibly detectable differences in electromigration profiles of proteins solubilized from permeabilized cells incubated with 32P NAD , the specific precursor of ADP-ribose. 3 There was an expected increase in ADPRT activity in HeLa cells exposed for up to 24 hours to the cancer- chemotherapeutic agent methotrexate. Methotrexate did not cause an increase in the rate of ADP-ribose degradation. The activation of the transferase is reflected in a general increase in protein ADP-ribosylation detected by autoradio- 32 graphy of P-labelled proteins in 6.25 - 18.25%T gradient acrylamide gels. There were three major acceptors with molecular masses of 17, 100 and over 100 kDa, which could be respectively a histone, a transferase-derived peptide fragment and the transferase itself. The enhanced protein ADP-ribosylation seen in the cells exposed to methotrexate is a direct consequence of drug-exposure, but does not have any significant influence over the course of events leading ultimately to cell death. 4 Determination of (ADP-ribose) degradation rates in permeabilized HeLa cells, measured as loss of acid- insoluble radioactivity from permeabilized cells previously incubated with 3H NAD , showed biphasic kinetics. The majority of label was lost within 20 min at pH 6.0 and 37 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. 5 DNA-cellulose column chromatography could not be used as a preliminary step in the purification at pH 6.5 of topoisomerase I. ADPRT was activated 3 fold in cells treated for 1 hour with 100u M DMS, as was topoisomerase I. Preliminary evidence is presented to suggest that topoisomerase I is ADP-ribosylated in control cells and is less modified in DNA-damaged cells, possibly by diversion of ADPRT activity to other acceptor proteins.