The long-term objective of the proposed research is to understand, at the molecular level, how D-glucose enters animal cells. The approach to this goal is the detailed elucidation of the structure and function of the glucose transporter from the human erythrocyte. There are four specific aims of the proposed research. First, the glucose transporter will be rigorously identified and completely purified. The present preparation of the transporter may contain 10 to 20% impurities, and it has been suggested that it is a proteolytic fragment from a larger protein. The methods to be used here are based upon the preparation of monoclonal antibodies against the transporter. In the course of this work, the nucleoside transporter from human erythrocytes will be largely purified. Second, the kinetic mechanism for transport will be completely determined. The rates of single turnovers of the purified, reconstituted transporter under various conditions will be measured by the stopped-flow method. Changes in intrinsic fluorescence of the protein upon ligand binding enable monitoring of these single turnovers. Third, the aminoacid sequence of the transporter will be obtained by isolating and sequencing the cDNA for the protein. This information, in conjunction with that from vectorial labeling studies in the laboratory, will be used to describe the folding pattern of this polypeptide in the erythrocyte membrane. Fourth, an attempt will be made to crystallize the transporter in a form suitable for x-ray diffraction analysis.
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