Figure 5.

Analysis of l-isoAsp damage in the native size-exclusion fractions of human whole-lens extracts by SDS-PAGE reveals that the HMW gel filtration fraction contains significant amounts of LMW l-isoaspartyl–containing species. Fractions from size-exclusion chromatography as in Fig. 4 were TCA-precipitated and resuspended in 100 mm Tris-HCl, pH 7.9, 150 mm NaCl, 0.1% SDS, 6 m urea, and l-isoAsp sites within the fractions were radiolabeled by PCMT1 and [³H]AdoMet as described in the legend to Fig. 3. Labeled fractions were then separated by SDS-PAGE on a 4–12% gradient gel. The Coomassie-stained gel is shown in the upper panels; the fluorograph (1-week exposure) is shown in the bottom panels. The bands corresponding to PCMT1 in the Coomassie-stained gel are indicated by asterisks. The migration positions of intact crystallins are indicated by brackets on the right-hand side of the gels. Labels above the lanes represent the corresponding peaks from the size-exclusion run (see Fig. 4). The age of lens sample in years is designated above the corresponding gel.