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Electrophoresis

Isoelectric focusing in immobilized pH gradients of the glucose transporter from human red cell membranes.


PMID 8137794

Abstract

Isoelectric focusing of the human red cell glucose transporter (a transmembrane protein) was performed in immobilized pH gradients. Isoelectric focusing of integral membrane proteins presents problems that are related to the amphiphilic nature of these proteins. Solubilizing additives must be used to counteract hydrophobic effects. In our case, urea and the nonionic detergent, Triton X-100, were used. Focusing was done at 15 degrees C. The isoelectric point (pI) of the glucose transporter (freshly purified by anion-exchange chromatography in the presence of octyl glucoside) was determined at 8.4 +/- 0.05 (n = 9), in good agreement with our earlier determinations by two-dimensional electrophoresis with isoelectric focusing in the presence of carrier ampholytes in the first dimension. The width of the focused zone was approximately 0.1 pH unit, more narrow than after focusing with carrier ampholytes. In an immobilized pH gradient from pH 7 to 10, the transporter region at pH 8.4 comprised one major and one or two minor zones. The pH interval 4-10 was also used and showed a single transporter zone. The glucose transporter tends to self-associate in detergent solution. Octyl glucoside-purified glucose transporter formed oligomers during incubation at 37 degrees C. Upon focusing, these oligomers appeared in a wide pH interval far below pH 8.4.

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T8532
Triton X-100, for electrophoresis