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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022996-10
Application #
3271451
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1976-09-01
Project End
1989-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
10
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Dartmouth College
Department
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Davies, A; Ciardelli, T L; Lienhard, G E et al. (1990) Site-specific antibodies as probes of the topology and function of the human erythrocyte glucose transporter. Biochem J 266:799-808
Baldwin, S A; Lienhard, G E (1989) Purification and reconstitution of glucose transporter from human erythrocytes. Methods Enzymol 174:39-50
Gould, G W; Lienhard, G E (1989) Expression of a functional glucose transporter in Xenopus oocytes. Biochemistry 28:9447-52
Appleman, J R; Lienhard, G E (1989) Kinetics of the purified glucose transporter. Direct measurement of the rates of interconversion of transporter conformers. Biochemistry 28:8221-7
Gould, G W; Lienhard, G E; Tanner, L I et al. (1989) Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters. Arch Biochem Biophys 268:264-75
Calderhead, D M; Lienhard, G E (1988) Labeling of glucose transporters at the cell surface in 3T3-L1 adipocytes. Evidence for both translocation and a second mechanism in the insulin stimulation of transport. J Biol Chem 263:12171-4
Froehner, S C; Davies, A; Baldwin, S A et al. (1988) The blood-nerve barrier is rich in glucose transporter. J Neurocytol 17:173-8
Allard, W J; Gibbs, E M; Witters, L A et al. (1987) The glucose transporter in human fibroblasts is phosphorylated in response to phorbol ester but not in response to growth factors. Biochim Biophys Acta 929:288-95
Horner, H C; Munck, A; Lienhard, G E (1987) Dexamethasone causes translocation of glucose transporters from the plasma membrane to an intracellular site in human fibroblasts. J Biol Chem 262:17696-702
Allard, W J; Lienhard, G E (1985) Monoclonal antibodies to the glucose transporter from human erythrocytes. Identification of the transporter as a Mr = 55,000 protein. J Biol Chem 260:8668-75

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