Figure 2.

The l-isoAsp modification accumulates to high levels within the aged human lens polypeptides, whereas endogenous methyltransferase repair activity is largely maintained with age. Lens extracts (25 μg of protein) were radiolabeled by 6 μg of PCMT1 and 10 μm [³H]AdoMet for 2 h at 37 °C as described under “Experimental Procedures.” A, l-isoAsp quantified in whole-lens extracts. Closed circles, a single eye lens; open circles, the other eye from the same individual. The solid line includes the WS extracts; the dashed line includes the urea-solubilized WI extracts. Each symbol represents one technical replicate. Error bars, S.D. of the biological replicates. Lines were linear regression fits in GraphPad, and the slope was found to be significantly nonzero for both WS and WI extracts at 0.0048 and 0.0045 p values, respectively. B, l-isoAsp levels quantified in dissected lens nuclear (red circles) and cortical (blue squares) extracts. The solid line and closed symbols represent the WS extracts; the dashed line and open symbols represent the WI extracts. Each symbol represents one technical replicate. Error bars, S.D. of the technical replicates. Lines were linear regression fits performed in GraphPad, and none of the lines had significantly nonzero slopes (p values were all >0.05). C, PCMT1 activity in whole-lens extracts was quantified by detecting the amount of [³H]AdoMet radioactivity transferred to 100 μm peptide substrate KASA(isoD)LAKY by 15 μg of lens extract protein in 2 h at 37 °C. Closed circles represent one lens from an individual; open circles represent the other lens from that individual. Each symbol represents one technical replicate. Error bars, S.D. of the technical replicates. In a linear regression fit performed in GraphPad, the slope of the line was significantly nonzero with a p value of 0.0258. D, PCMT1 activity was quantified in dissected lens nuclear (red circles) and cortical extracts (blue squares). Open and closed symbols represent each lens from one individual at 47 and 69 years old. Each symbol represents a technical replicate. Error bars, S.D. of the technical replicates. Lines were linear regression fits performed in GraphPad, and neither of the lines had significantly nonzero slopes (p values were greater than 0.05).