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CR Fleschner; Rat Lens Membrane Distribution of Spectrin and Ankyrin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):459.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the relative distribution of the cytoskeletal associated proteins spectrin and ankyrin among membrane fractions isolated from rat lenses. Methods: Lenses from 20 day old rats were removed, decapsulated, homogenized and separated into water soluble and water insoluble (WIF) fractions by differential centrifugation. The density of the water soluble fraction was increased to 1.23 g/ml with KBr and subjected to centrifugation, resulting in a floating membrane fraction (the non sedimenting membrane fraction, NSMF), a pellet (the cytoskeleton enriched fraction), and the water soluble fraction (WSF). Other membrane fractions were obtained from the interfaces of discontinuous sucrose gradient centrifugation of the WIF (25/45, 45/50, and ≷=50). Membrane fractions, and the cytoskeleton enriched fractions were extracted with 8 M urea and the urea soluble proteins were separated by SDS PAGE. Spectrin and ankyrin were identified and quantitated by western blotting. Results: Neither spectrin, nor ankyrin were detected in the WSF, and only trace amounts of spectrin and no ankyrin were found associated with the cytoskeleton enriched fraction. Surprisingly, the NSMF, which has been shown to possess the greatest relative concentration of intermediate filament proteins among the membrane fractions, also contained only trace amounts of spectrin and no ankyrin. The greatest relative concentrations of spectrin and ankyrin were found associated with the quantitatively major membrane fraction isolated by sucrose density centrifugation, with lesser amounts associated with the other membrane fractions isolated from the sucrose gradient. Conclusion: These results suggest that the cytoskeleton - plasma membrane complex associated with the NSMF is organized differently than that of the rest of the plasma membrane.
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