1) We plan to focus our studies on hexose transport by intestinal cells with the specific aims at the identification, isolation, purification and characterization of two major hexose carriers. These are the Na+-stimulated hexose carrier present in the microvilli and the facilitated Na+-independent carrier associated with basolateral membranes. The intestinal cells will be derived from rat intestinal mucosa and two intestinal tissue culture lines: (1) IEC cells, with characteristics of undifferentiated crypt cells; and (2) CaCo-2 cells which, depending on growth conditions, exhibit either undifferentiated or differentiated characteristics. We plan to prepare high purified brush border and basolateral membranes using immunoaffinity purification with antimembrane monoclonal antibodies. The identification of the hexose carriers will involve photoaffinity labeling and/or photo-reactive cross-linking agents. The transport function of the purified and isolated carriers wil be assessed by means of membrane reconstitution. 2) We will examine factors regulating and influencing hexose transport by exogenous factors such as antibodies, lectins and hormones and also the effect of in vitro growth conditions and nutrient supply. We will also examine the role of translation and transcriptional control on the regulation of hexose carrier synthesis and degradation/inactivation.
