Ethidium bromide-stained electrophoretic gels containing the smaller and larger ribozyme constructs exposed to varying radiation levels are seen in Fig. 2 a and c, respectively. In Fig. 2a, one prominent band corresponds to the transcribed 262-nt ribozyme. This band progressively decreases in intensity with increasing radiation exposure. No distinct band was detected at radiation doses greater than 36 Mrads. To control for the possibility of changes in ethidium bromide staining efficiency of irradiated ribozymes, 33P-labeled 262-nt ribozymes were irradiated, electrophoresed, and autoradiographed; there was no difference in the patterns of irradiated radiolabeled ribozyme as compared to ethidium bromide-stained nonradioactive ribozyme samples (data not shown). An indistinct smear was seen below the main band in all samples except the unirradiated control (Fig. 2 a and c). The smears shift from a less electrophoretically mobile form to a more rapidly migrat ing pattern with increasing doses of radiation (Fig. 2 a and c). The intensity of the major (78.5 kDa) band in Fig. 2a was evaluated by densitometry and is plotted as a function of radiation exposure (Fig. 3). The slope of this inactivation curve was used to calculate an estimated molecular mass of 80.1 kDa (Table 1). Because of the technical difficulties in obtaining quantitative data from gel stains, there is considerable scatter among the experiments, which is reflected in the large errors.
The larger ribozyme was irradiated under identical conditions and analyzed in the same manner (Fig. 2c). The ethidium bromide-stained band at 368 kDa decreased in intensity very rapidly after progressive radiation exposures (Fig. 2c). From these data, a target size of 319 kDa was calculated (Table 1). Ribozyme Activity. Equal amounts of ribozyme from each irradiated sample were reacted with a large excess of labeled substrate (E:S = 1:10). The reaction was stopped and the samples electrophoresed. Sample autoradiographs of gels for the smaller ribozyme (Fig. 2b) and the larger ribozyme (Fig. 2d) are shown. The labeled bands of the original substrate (314 nt in length) and two cleavage products of the expected lengths of approximately 100 and 214 nt are seen. The most intense product bands are seen in the control lane containing unirradiated ribozyme plus substrate. When incubated with equal amounts of substrate, equimolar amounts of ribozymes treated with progressively larger doses of radiation generate bands of decreasing intensity representing a reduced level of ribozyme activity (Fig. 2 b and d). The amount of uncleaved substrate and product bands were counted by 13-scanning the dried gels as described. After subtracting for background, the amount of product was calculated as a percentage of total counts loaded per lane, normalized to the control lane, and plotted as an inactivation curve with control representing full activity of the unirradiated ribozyme (Fig. 3). Based on the inactivation of cleavage activity, a target size is calculated as 14.8 kDa for the smaller ribozyme transcript (actual mass = 78.6 kDa). The larger ribozyme transcript showed a very slow decrease in cleavage activity after massive radiation exposure (Fig. 4). These data yielded a target size of 15.9 kDa (actual mass = 368 kDa) (Table 1), which is indistinguishable from that obtained with the smaller ribozyme.
The Km of the ribozyme reaction was measured for the small ribozyme transcripts in unirradiated samples and in samples exposed to 48 Mrads. No significant difference in Km values were observed, indicating that the loss in activity was due to a decrease in vmax·
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