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Allosterism and Na++-d-glucose cotransport kinetics in rabbit jejunal vesicles: Compatibility with mixed positive and negative cooperativities in a homo- dimeric or tetrameric structure and experimental evidence for only one transport protein involved

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Summary

We first present two simple dimeric models of cotransport that may account for all of the kinetics of Na++-d-glucose cotransport published so far in the small intestine. Both the sigmoidicity in the Na++ activation of transport (positive cooperativity) and the upward deviations from linearity in the Eadie-Hofstee plots relative to glucose concentrations (negative cooperativity) can be rationalized within the concept of allosteric kinetic mechanisms corresponding to either of two models involving sequential or mixed concerted and sequential conformational changes. Such models also allow for 2 Na++∶ 1 S and 1 Na++∶ 1 S stoichiometries of cotransport at low and high substrate concentrations, respectively, and for partial inhibition by inhibitors or substrate analogues. Moreover, it is shown that the dimeric models may present physiological advantages over the seemingly admitted hypothesis of two different cotransporters in that tissue. We next address the reevaluation of Na++-d-glucose cotransport kinetics in rabbit intestinal brush border membrane vesicles using stable membrane preparations, a dynamic approach with the Fast Sampling Rapid Filtration Apparatus (FSRFA), and both nonlinear regression and statistical analyses. Under different conditions of temperatures, Na++ concentrations, and membrane potentials clamped using two different techniques, we demonstrate that our data can be fully accounted for by the presence of only one carrier in rabbit jejunal brush border membranes since transport kinetics relative to glucose concentrations satisfy simple Michaelis-Menten kinetics. Although supporting a monomeric structure of the cotransporter, such a conclusion would conflict with previous kinetic data and more recent studies implying a polymeric structure of the carrier protein. We thus consider a number of alternatives trying to reconcile the observation of Michaelis-Menten kinetics with allosteric mechanisms of cotransport associated with both positive and negative cooperativities for Na++ and glucose binding, respectively. Such models, implying energy storage and release steps through conformational changes associated with ligand binding to an allosteric protein, provide a rational hypothesis to understand the long-time debated question of energy transduction from the Na++ electrochemical gradient to the transporter.

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Authors and Affiliations

  1. Membrane Transport Research Group, Department of Physiology, Faculty of Medicine, University of Montreal, H3C 3J7, Montreal, Quebec, Canada

    Catherine Chenu & Alfred Berteloot

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  1. Catherine Chenu
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  2. Alfred Berteloot
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This research was supported by grant MT-7607 from the Medical Research Council of Canada. One of the authors (A.B.) was supported by a scholarship from the “Fonds de la Recherche en Santé du Québec” and C. C. was supported by a fellowship from the GRTM. The technical assistance of Mrs. C. Leroy has been greatly appreciated. The authors also thank D.D. Maenz and C. Malo for insightful discussions and C. Gauthier for the art work.

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Chenu, C., Berteloot, A. Allosterism and Na++-d-glucose cotransport kinetics in rabbit jejunal vesicles: Compatibility with mixed positive and negative cooperativities in a homo- dimeric or tetrameric structure and experimental evidence for only one transport protein involved. J. Membarin Biol. 132, 95–113 (1993). https://doi.org/10.1007/BF00239000

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