It has long been established that the """"""""active transport"""""""" of sugars, amino acids, and other metabolites is due to the presence of Na gradients across plasma membranes and Na-cotransport systems in the membranes. Recently, the intestinal brush border Na/glucosecotransporter has been identified using fluorescent covalent probes bound to the glucose and/or Na-active sites. Our long term goal is to determine the structure of the Na/glucose cotransporter and to correlate this structure with the transport properties of the polypeptide(s).
The specific aims are: 1) isolate and purify the functional cotransporter to homogeneity. This will be accomplished by fluorescently labeling the glucose carrier covalently at the glucose and/or Na-binding sites, and following the enrichment of the protein through chromatofocusing and affinity chromatography; 2) determine the structure of the purified carrier (sugar content, amino acid composition, and amino acid sequences of the Na and glucose active sites); 3) determine the molecular topography of the active sites using fluorescent probes (fluorescent quenching, energy transfer, and native trypophan fluorescence); and 4) develop a structural and kinetic model of Na/glucose cotransport. Transport activity in highly purified brush border vesicles, soluble protein, and reconstituted systems will be measured using Na-dependent a) glucose transport, b) phlorizin binding, c) potential changes, and d) quenching of fluorescent probes on the glucose-site. These studies will have general impact on our understanding of the mechanism of sugar, amino acid, metabolic intermediate transport across plasma membranes of the intestine, liver, kidney and brain, and particular relevance to the absorption of sugars by the small intestine.
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