Age-related increase in concentration and aggregation of degraded polypeptides in XXXX lenses.

@entity1 @entity12360 @entity1133
Proteins from @entity1 lenses of various ages were separated into three fractions based on their solubility; @entity12360 ( @entity12360 ), water-insoluble- @entity1133 -soluble (WI-US) and water-insoluble- @entity1133 -insoluble (WI-UI). The SDS-PAGE of these fractions showed increasing quantities of degraded polypeptides (mol. wt; Mr less than 18 kDa) with aging. On separation of degraded polypeptides from the rest of lens crystallins by gel-filtration chromatography under denaturing conditions, the polypeptides showed an age-related concentration increase in the @entity12360 and WI-US fractions, which constituted 12-14% and 17-18% of the total proteins respectively. The degraded polypeptides from WI-US fractions of lenses from 50-yr-old donors exhibited five polypeptides ranging in Mr between 3- and 18 kDa. The degraded polypeptides isolated from @entity12360 and WI-US fractions of lenses of various ages self-aggregated on storage to protein moieties with Mr ranging between 18- and greater than 1500 kDa. An antiserum was raised against degraded polypeptides isolated from WI-US protein fraction of 50-yr-old @entity1 lenses. During the Western blot analyses, the antiserum exhibited specific immuno-reaction with alpha-crystallin and 20-kDa gamma-crystallin species and with degraded polypeptides among various @entity1 lens crystallins tested. Similarly, when the self-aggregated degraded polypeptides of @entity12360 and WI-US fractions were reacted with the antiserum, several polymeric proteins of increasing Mrs were observed. In addition, when the heavy molecular weight (HMW) proteins from @entity12360 proteins and the total WI proteins of lenses of various ages were probed with the antiserum, several polymeric proteins, similar to the in vitro self-aggregated polymers of degraded polypeptides as previously described, were seen. Such polymers were in relatively lower quantities in the total @entity12360 proteins of lenses of various ages. These results suggest an apparent age-related polymerization of degraded polypeptides into HMW proteins leading to their insolubilization.
