In vitro glycation and acetylation (by aspirin) of rat crystallins

Abstract

In vitro studies with rat lens crystallins were conducted to explore the mechanism by which aspirin (ASA-acetylsalicylic acid) could inhibit cataractogenesis. The purpose of the present study is to show whether γ-crystallin is the primary target for glycation by glucose and acetylation by ASA. Lens soluble fractions from one and seven month old Sprague-Dawley rats were incubated with 5 mM [ 14C]glucose with and without 10 mM ASA. α, β, and γ-crystallins were separated by molecular sieve HPLC and specific activities of each crystallin determined. In vitro acetylation was also studied by measuring protein bound [ 14C]acetyl groups after incubation with [ 14C]acetyl ASA. There was 2 to 4-fold faster glycation of γ-crystallin than all other crystallins from 1-month-old rats and ASA inhibited glycation of γ-crystallin four times more than that of α and β-crystallins, thus showing preferential glycation of γ-crystallin and its selective inhibition by ASA. [ 14C]acetyl incorporation showed increased acetylation of γ-crystallin in one month old rats, whereas in older lenses acetylation of other crystallins predominated. Treatment with 10 mM ASA showed 35% decrease in free -NH 2 groups but protein thiols remained unchanged.