This research program is a continuous effort to isolate and reconstitute the structural unit of glucose transport carrier function of human erythrocytes, and to characterize its operation at the molecular level. To this end, we propose the following specific points to be studied: 1) To characterize the """"""""180,000 dalton-membrane protein,"""""""" which we have differentially labeled by fluorodinitrobenzene and tentatively proposed to be glucose carrier. This would include an isolation on a preparative scale; chemical and physical characterization; and reconstitution of the carrier activity in in vitro membrane structures from the protein. 2) To isolate and characterize the """"""""glucose-sensitive, cytochalasin B binding protein,"""""""" which we have also tentatively proposed to be the carrier, by means of covalent labeling or affinity chromatography. This would involve synthesis of protein-reactive cytochalasin B derivatives as covalent reagents; structure-activity relationship studies on cytochalasin B and the derivatives, both in terms of the glucose-carrier inhibition and of the glucose-sensitive binding; isolation and identification by covalent labeling and/or affinity chromatography; and characterization of the binding protein in terms of the glucose carrier activity in a reconstituted in vitro system. 3) To study the mode of information-transduction from insulin-receptor to glucose carrier, in native and in model membrane systems. This would include isolation of the insulin receptors; attempts at isolation of """"""""180,000 dalton-protein"""""""" and """"""""glucose-sensitive, cytochalasin B binding protein,"""""""" both from fat cell membranes; and subsequent characterization of interaction of these proteins with insulin receptors.
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