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Margaret H. Garner, Yongli Kong; Lens Epithelium and Fiber Na,K-ATPases: Distribution and Localization by Immunocytochemistry. Invest. Ophthalmol. Vis. Sci. 1999;40(10):2291-2298.
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purpose. To use immunofluorescence and immunogold techniques to identify the
catalytic subunits of the Na,K-ATPases of the lens and to determine
their location in the cells of the epithelium and cortex of bovine and
methods. Frozen sections of capsulated and decapsulated bovine and human lenses
were prepared, blocked, and treated with affinity-purified polyclonal
rabbit antibodies to the Na,K-ATPase catalytic subunit isoforms with
subsequent treatment with fluorescein isothiocyanate–labeled goat
anti-rabbit IgG and visualization of the fluorescence by light
microscopy. An immunogold-labeled goat anti-rabbit IgG was used to
detect, by electron microscopy, the binding of the same
affinity-purified polyclonal antibodies to thin sections of
decapsulated lenses that had been fixed and embedded in Lowicryl K4M.
The results were confirmed by staining of western blot analysis of
sodium dodecyl sulfate–polyacrylamide gel separations of enriched
membrane preparations from bovine and human lenses.
results. The three common catalytic subunits of the Na,K-ATPases are present in
the plasma membranes of lens epithelium, lens fibers, or both. The data
indicate a polarized distribution of the α1 and α3 catalytic
subunit isoforms in central epithelium. In the cortical fibers, theα
2 isoform is present around the interdigitations. The α3 isoform
is found in the interdigitation-free regions of human cortical fibers.
conclusions. This unique distribution of Na,K-ATPases precludes the popular
pump-leak model for lens monovalent cation homeostasis. The functional
significance of the distribution of Na,K-ATPases in the lens epithelium
and superficial fibers is currently under
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