A New Model for Assessing Proteolysis in the Intact Mouse Lens in Organ Culture

Abstract

Purpose: To develop a new method to investigate proteolysis in the intact lens in organ culture. Methods: Intact mouse lenses were assayed at regular intervals for proteolytic activity using fluorogenic peptide substrates ± addition of ionomycin. Specific inhibitors were used to determine the activity of calpains, the proteasome and acid lysosomal enzymes. Results: Significant levels of proteolytic activity were present in the intact lens. Proteolysis was stimulated by ionomycin. Preincubation with an inhibitor to the proteasome significantly decreased proteolysis whereas inhibitors of calpain and acid lysosomal enzymes did not. Conclusion: This study indicates that in the intact mouse lens in culture, the proteasome is an important protease. Its activity is at least partially regulated by calcium.

Changes in Activity and Kinetic Properties of the Proteasome in Different Rat Organs during Development and Maturation

“…This is supported by findings from our group indicating that intracellular proteasome activity in human lens epithelium may be stimulated by calcium both from external and internal stores [28]. Previous findings from our group also demonstrate that the chymotrypsin-like activity of the proteasome in intact mouse lens can be stimulated by increased intracellular Ca 2+ -levels [29]. The present study is the first to demonstrate that calcium-stimulated chymotrypsin-like activity of the proteasome decreases with a transition from sexually immature (7 days old) to mature/fertile (2–4 months) animal.…”

A dominant vimentin mutant upregulates Hsp70 and the activity of the ubiquitin-proteasome system, and causes posterior cataracts in transgenic mice

“…For proteasome analysis, 6 dissected lenses or a confluent cell layer from one 10 cm cell culture dish were homogenized at 4°C for 10 minutes in 2 ml Earle's balanced salt solution (EBSS) using a Potter-Elvehjem homogeniser (Braun, Germany) at 1200 rpm . Cleavage of the synthetic proteasome substrate III (Calbiochem) was measured with a Tecan Genios Spectrafluor Plus multititer plate reader (Tecan, Germany) as described previously (Petersen et al, 2004).…”

The Proteasome and Intracellular Redox Status: Implications for Apoptotic Regulation in Lens Epithelial Cells

“…Proteolysis in intact mouse lenses was measured essentially as described in Petersen et al 23 Because the LLVY-substrate is the only cell permeable of the three substrates used in this study, only the chymotrypsinlike activity was investigated in whole lenses. Prior to the assay, the lenses were transferred to a white 96-well plate with transparent bottom containing 100 µl EBSS.…”