The unidirectional influx of alpha-methylglucoside (alpha-MG) by isolated chicken intestinal epithelial cells is 98% inhibited by phlorizin. The remaining 2% of the total influx occurs in the absence of Na+, is not sensitive to phloretin, and is equal to the diffusional entry rate for 2-deoxyglucose. The glucoside is much more strongly accumulated (75-fold) than 3-O-methylglucose (3-OMG) (10-fold). Inhibitors of the serosal sugar carrier (phloretin, cytochalasin B, theophylline, and flavanoids) do not enhance alpha-MG accumulation. It is concluded that the glycoside is not a substrate for the intestinal serosal transport system. Steady-state gradients of the sugar can be represented accurately by a concentrative, phlorizin-sensitive system that is opposed by a diffusional efflux process.
To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by x-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Initial purifications were based on expression of the genes under control of a galactose-inducible promoter, but higher cell densities and improved expression have been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification-some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. The size, oligomeric state, and composition of purified protein-detergent complexes purified under different conditions were analyzed using a colorimetric assay of detergent concentration and by analytical size exclusion chromatography using static light scattering, refractive index detection, and UV absorption to monitor the elution profiles. Effective procedures were developed for obtaining high concentrations of purified TMPs without excessively concentrating detergents.
Sodium-dependent sugar transport systems involve the function of membrane components that couple the transmembrane flow of Na+ to the concomitant flow of certain sugar molecules. The coupling stoichiometry between Na+ and sugar fluxes via these systems must be measured under conditions in which the membrane potential does not change due to the induction of transport or during the interval of flux measurement. This can be accomplished by utilizing gradients of highly permeant ions (NO-3 and K+ plus valinomycin) to create diffusion potentials of sufficient magnitude that the sugar-induced Na+ flux does not introduce an appreciable change in the imposed potential. Under these conditions, the coupling stoichiometry for chicken intestinal cells proves to be 2 Na+:1 sugar as reported earlier for studies performed in the absence of a membrane potential. When control of the potential is not maintained, a coupling ratio of 1:1 is observed. The stoichiometry does not change as a function of Na+ concentration, which suggests that carrier forms with only one Na+ bound do not contribute to the carrier-mediated Na+ or sugar fluxes. When no potential is present, the stoichiometry is modified by the level of intracellular Na+ and sugar in a manner indicative of a transport mechanism in which Na+ must dissociate from the "loaded" carrier at the inward facing membrane surface before the sugar molecule dissociates.
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