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The heritable sheep cataract : a model with which to investigate calpain involvement in cataractogenesis

Research Archive

The heritable sheep cataract : a model with which to investigate calpain involvement in cataractogenesis

Clapham, M. J.
Type: Thesis
Permalink: https://hdl.handle.net/10182/2489
Originally published: 1997 by Lincoln University
Keywords: cataract; m-calpain; calpastatin; lens; crystallins; proteins; assay; chromatography; sheep; electrophoresis

Abstract:

Calpains represent a family of non-lysosomal calcium-dependent proteases. Lenses from a variety of animals have been shown to contain calpain and its endogenous inhibitor, calpastatin. During cataractogenesis calcium levels in lens tissue are observed to increase sufficiently to activate calpain in a variety of animal models. It is therefore, postulated that calpain is activated during cataractogenesis, resulting in proteolysis and insolubilisation of crystallins, the proteins responsible for lens transparency. The aim of this thesis was to utilise the heritable sheep cataract to investigate the role of calpain in cataractogenesis. A line of sheep displaying the heritable cataract phenotype were bred and cataract development described. Calpain, calpastatin and the crystallins were separated from both normal and diseased lenses using ion-exchange chromatography. Calcium concentrations were determined using Atomic Absorption Spectroscopy. A pre-existing FTC-casein fluormetric assay was modified to increase its sensitivity and permit the measurement of calpain and calpastatin activity from sheep lenses. Crystallins from normal and cataract lenses were separated on SDS-PAGE, as were crystallins incubated in vitro with purified m-calpain. Proteolytic changes observed with cataractogenesis were compared to those resulting from in vitro proteolysis by calpain. Lenses were found to contain m-calpain and calpastatin. Furthermore, during the development of cataracts, activity of m-calpain was reduced dramatically, while calpastatin activity persisted. This may be due to either autolysis of calpain, or its association with heplasma membrane. Proteolysis of α-, βH and βL-crystallin, but not γ-crystallin, was observed in vivo and in vitro. Fragments resulting from proteolysis were examined on SDS-PAGE and were comparable. This evidence supports the hypothesis that calpain induced proteolysis of lens crystallins is part of the mechanism involved in the development of cataracts. This thesis was successful in characterising a new model with which to study the involvement of calpain in cataractogenesis. This may be used to study the aeitology of cataract development and to investigate drug therapies designed to slow or prevent cataract formation.

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