Ribose electrophoretic mobility

The unknown phosphate ester had the same electrophoretic mobility as 2-deoxy ribitol 5-phosphate and it seemed reasonable to expect that in the conditions used (0.1 M pyridinium acetate buffer of pH 5) 2-deoxy ribitol-4- and -5-phosphates would behave similarly therefore it was considered probable that the unknown phosphate ester is 2-deoxy ribitol 4-phosphate, resulting from the reduction of the periodate resistant 2-deoxy ribose 4-phosphate. However, the possibility that both 2-deoxy ribitol 4-phosphate and 2-deoxy erythritol 3-phosphate (formed from... 

These results suggest that the three particular proteins identified are modified by only short ADP-ribose chains and that their greater radioactive content is because a larger proportion of the molecules in each population becomes modified in the methotrexate-treated cells. Longer ADP-ribose chains would lead to clearly detectable changes in electrophoretic mobility as a result of the increased mol. wt. 

HS-C protein is poly(ADP-ribose) polymerase. To test this possibility further, the protein was subjected to ADP-ribosylation with [i C]NAD and nicked DNA. The reaction product was subjected to SDS-PAGE. Most of the radioactivity was located near the origin or at a position that approximately corresponded to the Coomassie blue-stained protein band in a parallel track (data not shown). Since poly(ADP-ribose) polymerase is capable of auto-ADP-ribosylation and this modification can alter electrophoretic mobility (6), the two radioactive peaks observed after ADP-ribosylation of HS-C protein probably correspond to the different levels of ADP-ribosylation occurring in the enzyme molecule. That the radioactive peaks were indeed generated by a specific enzymatic reaction was shown by the lack of incorporation of [i C]NAD at 4°C or in the absence of nicked DNA (data not shown). 

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