Purchase this article with an account.
M.S. Muir, J.D. Morton, L.J. G. Robertson; Calpain II Induces Insolubilisation of Ovine Lens Crystallins in vitro and These Insoluble Proteins May Induce Cataract in vivo . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3882.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if incubation of ovine lens proteins with calpain II results in insolubilisation of the crystallins Methods: Calpain II was purified from sheep lung by applying the soluble fraction to a combination of chromatography columns. Calpain II activity was measured and the protein concentration determined. Lenses were dissected from lamb eyes, decapsulated and homogenised. The pellet was washed twice then resolubilised. Protein concentrations were determined for the pellet and supernatant. The soluble lens crystallins were incubated with the purified calpain II in the presence of 3 mM Ca2+ at 37°C for 2 hours. The reaction was terminated with the addition of EGTA centrifuged and the supernatant and pellet collected. The pellet was then resolubilised by sonication. Lenses were also obtained from lamb eyes with cataracts and the insoluble protein isolated from the cortex as above. Calpain II induced changes were compared to the insoluble fraction of the cataract lenses using two dimensional electrophoresis (2DE). Results: The crystallin profile from the insoluble cortex fraction of cataractous ovine lenses contained large numbers of proteolysed α and ß crystallins. These crystallins were not present in the insoluble fraction of the cortex from a normal lens. Incubation of soluble lens proteins with exogenous calpain II in the presence of Ca2+ resulted in the production of water–insoluble proteins. The proportion of insoluble protein produced using 3mM Ca2+ was 6% of the soluble protein. Protein insolubilisation was not detected in the presence of 10mM EGTA or when calpain II was omitted. When visualised many proteins produced by calpain II incubation were not found in the insoluble protein fraction from normal lenses. The insoluble proteins produced by incubation with calpain II were similar to the insoluble proteins present in the cortex from cataractous ovine lenses. Conclusions: This experiment suggests that calpain II is at least partially responsible for the insolubilisation of protein ovine cataractous lenses. Further experiments will determine exact truncation sites on insoluble proteins to positively identify the calpain proteolytic signature.
This PDF is available to Subscribers Only