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C.R. Fleschner; Lens Membrane Distribution Of Active Calpains . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4099.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the distribution of calpain activities among membrane fractions isolated from the cortex and nucleus of rat lens.
Decapsulated rat lenses were separated into cortex and nucleus by gentle stirring in buffer containing a protease inhibitor cocktail. Homogenates of the separated lens regions were spun at 20 000xg for 30m to sediment the water insoluble fraction, and membrane fractions were isolated from the water insoluble fraction by discontinuous sucrose density centrifugation. The non sedimenting membrane fraction was isolated from the supernatant of the first homogenate spin by under laying with a cushion of 50% sucrose (w/w), and spinning the tube at 100 000xg overnight. The membrane fraction was recovered from the interface of the supernatant and the 50% sucrose. Calpain activity was examined by native gel zymography, using casein as the substrate.
Casein zymography detected activity attributable to m–calpain (calpain 2), µ–calpain (calpain 1) and Lp82/85. M–calpain activity was restricted to the water soluble fraction and was detected in both the cortex and nucleus, with a greater specific activity in the cortex. µ–Calpain activity was also restricted to the water soluble fraction, and was almost exclusively detected in the cortex; only a trace amount of activity was detected in the nuclear water soluble fraction. Lp82/85 activity appeared to be restricted to the water soluble fraction in the cortex, but was found in both the water soluble and water insoluble fractions of the nucleus. The membrane associated Lp82/85 activity of the nucleus was restricted to the cytoskeletal enriched fraction and the two densest membrane fractions; essentially no Lp82/85 activity was associated with the lighter, major membrane fraction.
The greater activity of Lp82/85 in the nucleus may be related to the development of dense nuclear cataracts associated with the selenite cataract model, which is thought to develop by activation of calpain activity. The absence of Lp82/85 activity in the major membrane fraction of the nucleus suggests that proteins associated with this fraction may be protected from calpain proteolytic activity.
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