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Partial purification and reconstruction of the Na+-D-glucose cotransport protein from pig renal proximal tubules

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Koepsell,  Hermann
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Menuhr,  Helmut
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Ducis,  Ilze
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Wissmüller,  Theo F.
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Koepsell, H., Menuhr, H., Ducis, I., & Wissmüller, T. F. (1983). Partial purification and reconstruction of the Na+-D-glucose cotransport protein from pig renal proximal tubules. The Journal of Biological Chemistry, 258(3), 1888-1894. doi:10.1016/S0021-9258(18)33072-2.


Cite as: http://hdl.handle.net/21.11116/0000-0007-EB1B-3
Abstract
Brush border membranes from renal proximal tubules were solubilized with deoxycholate, and the proteins were incorporated into liposomes formed from cholesterol and phospatidylserine by a freeze-thaw procedure. In the proteoliposomes Na+-D-glucose cotransport was demonstrated by showing that the D-glucose concentration in the liposomes increased far above the equilibrium value if a Na+ gradient was applied. The initial D-glucose uptake rate, stimulated by an inside directed gradient of 89 mM Na+, was 4 pmol/mg of protein-1 s-1. High affinity phlorizin binding could not be measured. After two precipitation steps with the solubilized membrane proteins, a protein fraction was obtained in which significantly high affinity phlorizin binding was detected. After reconstitution, proteoliposomes were formed in which more than 70% of the protein was represented by two polypeptides with molecular weights of 94,000 and 52,000. An initial Na+ gradient-dependent D-glucose uptake rate of 118 pmol/mg of protein-1 s-1 was obtained. In these liposomes, the D-glucose uptake rate could be inhibited by phlorizin (Ki = 0.3 μM), and 55-pmol phlorizin-binding sites per mg of protein (KD = 0.5 μM) were measured. In different liposomal preparations a correlation between Na+ gradient-dependent D-glucose uptake rate and the amount of 52,000 molecular weight polypeptide was observed.