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J. K. Whitman, C. R. Fleschner, M. K. Stuart; Isolation of a Ganglioside Unique to the Water-Insoluble Fraction of Ocular Lens. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4607.
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The lens fiber cell plasma membrane is organized into domains with compositions that differ from the majority of the lipid bilayer. These domains may impart specialized functions such as cell-cell adhesion or nutrient transport. In prior studies, we isolated two fiber cell membrane fractions thought to represent distinct lens domains: the 25/45 fraction isolated by discontinuous sucrose density centrifugation from the water-insoluble fraction of rat lens homogenates, and the non-sedimenting membrane fraction (NSMF) collected from atop the water-soluble fraction. Our long-term goal is to define the biochemical differences between the 25/45 fraction and NSMF to support the hypothesis that these fractions represent different lens domains. In this study, our specific aim was to identify the antigen recognized by monoclonal antibody (Mab) 10A5, which reacts exclusively with the 25/45 fraction but not with the NSMF.
The antigen recognized by Mab 10A5 and proteins physically associated with the antigen were isolated from the fraction by immunoprecipitation. Proteins were identified by SDS-PAGE, Western blotting, and MALDI-ToF mass spectroscopy. Lipids of the fraction were extracted by the Folch method and analyzed by thin-layer chromatography.
The antigen reactive with Mab 10A5 was identified as a ganglioside by Folch extraction of the 25/45 fraction, followed by thin layer chromatography and resorcinol staining of the immunoreactive spot. Immunoprecipitation of the ganglioside from the 25/45 fraction resulted in the co-precipitation of both galectin related inter-fiber protein (GRIFIN) and alphaA-crystallin, two important structural proteins within the ocular lens.
Our results suggest that the ganglioside, GRIFIN, and alphaA-crystallin exist as a complex within the fiber cell plasma membrane. Furthermore, the ganglioside appears to be unique to the 25/45 fraction, supporting the hypothesis that the 25/45 fraction and NSMF comprise distinct lens domains.
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