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C.R. Fleschner; Active Lens Specific Calpain (Lp82/85) Is Associated With the Non–sedimenting Membrane Fraction . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1870.
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Purpose:. To isolate the non sedimenting membrane fraction so as to be able to detect calpain activity, and to examine the distribution of calpain among membrane fractions isolated from rat lens. Methods: Homogenates of decapsulated rat lenses 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. The abundance of Lp82/85 protein was examined by western blot. Results: A small membrane fraction comprising about 6% of the total membrane MIP was recovered from the interface of the first 20 000xg supernatant and the 50% sucrose. This membrane fraction was enriched in MIP by at least 14 fold over the homogenate. Casein zymography detected activity attributable to m–calpain, µ–calpain and Lp82/85. M–calpain and µ–calpain activities were restricted to the water soluble fraction. Lp82/85 activity was detected in the water soluble fraction and in the non sedimenting membrane fraction, with perhaps trace amounts in the other membrane fractions. The amount of immunoreactive Lp82/85 found by western blot in the non sedimenting membrane fraction was almost twice as much as found in the major membrane fraction (which accounted for about 35% of the total membrane MIP). Conclusions: 1.) The non sedimenting membrane fraction may be isolated from the supernatant of the first 20 000xg spin of the homogenate by high speed sedimentation over a cushion of sucrose. 2.) Active lens specific calpain (Lp82/85) is associated with the non sedimenting membrane fraction. 3.) The non sedimenting membrane fraction is enriched with immunoreactive Lp82/85.
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