Kinetics of the purified glucose transporter. Direct measurement of the rates of interconversion of transporter conformers
- 1 October 1989
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 28 (20), 8221-8227
- https://doi.org/10.1021/bi00446a038
Abstract
There is considerable evidence that the mechanism of glucose transport by the transporter of human erythrocytes is one in which the transporter oscillates between two conformations, To and Ti. Each conformer possesses a single glucose binding site that in vivo faces either the extracellular space (conformer To) or the cytoplasm (conformer Ti). In this study, the interconversions of these confomers in the absence and presence of D-glucose have been directly observed by means of the stopped-flow method with fluorescence detection. Nearly unidirectional conversion of one conformer to the other was accomplished by rapidly mixing purified transporter (a mixture of To and Ti) with either 4,6-ethylidene-D-glucose, which preferentially binds to To, or phenyl .beta.-D-glucoside, which preferentially binds to Ti. The values of the individual rate constants for the conversion of Ti to To and vice versa in the absence and presence of D-glucose at 10.0.degree. C have been obtained, and these show that the kinetics are consistent with the alternating conformation model for transport. Conformational change occurs much more rapidly with glucose bound to the transporter. Furthermore, the activation energy Ea for conformer interconversion is much less when glucose is bound than for unliganded transporter. For example, Ea is approximately 28 kcal/mol for Ti .fwdarw. To versus 17 kcal/mol for Ti + S .fwdarw. ToS, where S is glucose. The .alpha.-anomer of glucose was 37% more effective than the .beta.-anomer in speeding the interconversion. Analysis of the fluorescence change upon mixing the transporter with several concentrations of 4,6-ethylidene-D-glucose revealed that the intrinsic fluorescence of the To form is less than that of the Ti form by 20% or more.This publication has 9 references indexed in Scilit:
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