Figure 3.
l-isoAsp damage accumulates with age throughout a range of polypeptide sizes in water-soluble extracts of human lenses, primarily within LMW species. WS extracts (25 μg of protein) were incubated with 0.3 μm [3H]AdoMet and with (+) or without (−) 5 μg of PCMT1 for 2 h at 37 °C, and polypeptides were analyzed by SDS-PAGE using a 12% acrylamide matrix. Coomassie-stained gels are shown at the top; fluorographs (2-day exposures) are shown at the bottom. Molecular weight markers (kDa) are indicated with arrows to the left of each gel and include myosin, β-gal, phosphorylase b, BSA, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme, and aprotinin (Bio-Rad SDS-PAGE Molecular Weight Standards, Broad Range, catalogue no. 161-0317). The band corresponding to PCMT1 is indicated by asterisks. The migration positions of intact crystallins are indicated by brackets on the right-hand side of the gels. A, whole-lens extracts from two lenses of each individual. B, nuclear and cortical separated extracts from a single lens (1-day exposure). C, densitometry of lanes from whole-lens sets 1 and 2. Error bars, S.D. of the two lanes. D, densitometry of lanes from nuclear and cortical sets.