Proteolytic cleavage of N-Succ-Leu-Leu-Val-Tyr-AMC by the proteasome in lens epithelium from clear and cataractous human lenses.
With ageing, accumulation of modified proteins occur in the lens, forming light scattering aggregates. The multicatalytic proteinase complex, or proteasome, is known to be the major system for removal of damaged proteins in many tissues. In this study we attempted to compare levels of proteasome activity in human lens epithelium from clear vs. cataractous lenses. Normal lenses were obtained from eye donors in a cornea bank and samples from cataractous lenses were obtained from an eye clinic during cataract surgery. Proteolytic activity was quantified using the synthetic peptide substrate N-Succ-Leu-Leu-Val-Tyr-AMC, a substrate often used to measure the chymotrypsin-like activity of the proteasome. Addition of 100 micron lactacystin, a proteasome specific inhibitor, totally inhibited proteolysis, certifying the specificity of the assay. Hydrolysis was detected over time as the appearance of the flourogenic cleavage product and correlated to the area of the epithelium-capsule specimens. Proteolytic cleavage of N-Succ-Leu-Leu-Val-Tyr-AMC by the proteasome was higher in lens epithelium from clear donor lenses as compared to samples from cataractous lenses. Median activity in the latter was only 19% of that in the former, a highly significant difference. There was no difference in activity of the proteasome when looking at cortical vs. non-cortical cataract, nor was there any difference between genders. Regression analysis did not reveal any age-dependent relationship, either in the clear group or in the cataractous group. This work is the first to show differences in proteasome activity between clear and cataractous lenses.
Andersson
M
M
Institute of Clinical Neuroscience, Department of Ophthalmology, Medicinaregatan 5, Göteborg, Sweden.
Sjøstrand
J
J
Karlsson
J O
JO
D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000378:metabolism; D002386:Cataract / Q000201:enzymology*; D003374:Coumarins / Q000378:metabolism*; D046508:Culture Techniques; D010450:Endopeptidases / Q000378:metabolism*; D004848:Epithelium / Q000201:enzymology; D005260:Female; D006801:Humans; D007908:Lens, Crystalline / Q000201:enzymology*; D008297:Male; D008875:Middle Aged; D009842:Oligopeptides / Q000378:metabolism*; D013379:Substrate Specificity
D003374:Coumarins; C114703:N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin; D009842:Oligopeptides; D010450:Endopeptidases