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A Petersen, M Andersson, J Sjöstrand, J-O Karlsson; Proteolytic Systems of the Mouse Lens. Effects of Ionophors and Growth Factors . Invest. Ophthalmol. Vis. Sci. 2002;43(13):462.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To characterize the basal proteolytic activity in the intact mouse lens in organ culture. To determine the effect of calcium ionophors, growth factors and insulin on proteolysis. Methods: Lenses from adult mice were placed in a medium containing a fluorogenic protease substrate. The preparation was placed in a fluorometer and continuously assayed for the degradation of the substrate during 4 hours. The following proteolytic systems were studied; calpain, the proteasome, metalloproteases, cathepsin B and cathepsin D/E. Results: The level of basal calpain and proteasome activity, as measured by degradation of LLVY-AMC, was low. This basal activity could not be inhibited by inhibitors of calpain (calpeptin), the proteasome (lactacystin) and the lysosomal enzymes (monensin). An increase of the proteolytic activity could be observed after application of thapsigargin (10µM) and the Ca-ionophore, ionomycin (10µM). This increase could be inhibited by the pretreatment with lactacystin. The growth factors, TGF-ß (50 ng/ml), FGF (50 ng/ml) and insulin (20 µg/ml) had no significant effect on the activity of calpain or of the proteasome during 24 hours. The proteolytic activity of metalloproteases was very low using non-cellpermeable substrate (AAA-AMC, DQ gelatine and Suc-Gly-Pro-Leu-Gly-Pro-MCA). Significant activity was obtained with a cell-permeable substrate (AAF-AMC and with MOCAc-Pro-Leu-Gly-Leu-A2Pr(Dnp)-Ala-Arg-NH2). Cathepsin B and D/E activity was relatively low. After the proteolysis assay, the morphology of the lenses was examined using metacarn fixation and plastic resin embedding. Conclusion: The basal proteolytic activity of the lens was relatively low. Increased Ca-levels increased the proteolytic activity, which may derive from the proteasome and possibly from the calpain system. The growth factors did not affect the proteolysis in the lens for up to one day. Some swelling of the outer cortical fibres was seen in ionomycin and thapsigargin treated lenses.
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