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R.F. Jacob, C.A. Day, R.P. Mason, R.J. Cenedella; Biochemical and Structural Characterization of Lipid Raft–Like Fractions Isolated From Bovine Lens Cortical Membrane by Octyl Glucoside Extraction . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1864.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:The lens fiber cell plasma membrane is a highly–ordered structure enriched in sphingomyelins, cholesterol and an assortment of proteins, including caveolin–1, a marker of lipid raft–like domains. These specialized membrane fractions were recently identified in human lens fiber cells by treating isolated plasma membranes with 1% Triton X–100. This approach may be limited, however, in isolating highly–ordered lipid domains from cells with tightly assembled membranes. The goal of this study was to characterize the composition and biophysical structure of lipid raft–like domains isolated from bovine lens fiber cell membranes by treatment with both Triton X–100 and octyl glucoside at high ionic strength, a technique recently used to extract raft–like membranes from trachealis smooth muscle. Methods: Lipid raft fractions were obtained from bovine lens cortex by treating total membrane with buffer containing 1% Triton X–100, 35 mM octyl glucoside and 600 mM NaCl and centrifuging the mixture for 20 hr in a continuous sucrose gradient. Sample fractions were collected from the gradient and prepared for protein and lipid quantitation. Aliquots of each fraction were subjected to SDS–PAGE analysis of caveolin–1 and main intrinsic protein (MIP 26). Component organization and structural properties of the lipid raft fractions were measured using small angle x–ray scattering (SAXS) approaches. Results: Treatment of lens membranes with Triton X–100 and octyl glucoside resulted in recovery of 25–30% of lens total membrane in the high–buoyancy (low density) fractions, which were enriched with caveolin–1, sphingomyelin and cholesterol. MIP 26 was present in the low density fractions, but was more abundant in the higher density fractions. SAXS analysis of the low density lipid raft fractions demonstrated the presence of two phases with periodicities of ∼33 Å and 60 Å, consistent with distinct cholesterol and phospholipid domains, respectively. A third phase, with a periodicity of ∼85 Å, was also detected, which we speculate is an annular lipid domain since it appeared sensitive to changes in temperature and relative humidity and was essentially removed by Folch extraction. Conclusions: Lipid raft–like domains of the bovine lens membrane contain high levels of cholesterol, sphingomyelin and protein, which are arranged into discrete microdomains. This study provides novel insights into the complex structural organization of the lens membrane as well as other tightly associated cellular membranes.
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