Kinetic analysis of mechanism of intestinal Na+-dependent sugar transport

Zero-trans kinetics of Na+-sugar cotransport were investigated. Sugar influx was measured at various sodium and sugar concentrations in K+-loaded cells treated with rotenone and valinomycin. Sugar influx follows Michaelis-Menten kinetics as a function of sugar concentration but not as a function of Na+ concentration. Nine models with 1:1 or 2:1 sodium:sugar stoichiometry were considered. The flux equations for these models were solved assuming steady-state distribution of carrier forms and that translocation across the membrane is rate limiting. Classical enzyme kinetic methods and a least-squares fit of flux equations to the experimental data were used to assess the fit of the different models. Four models can be discarded on this basis. Of the remaining models, we discard two on the basis of the trans sodium dependence and the coupling stoichiometry [G. A. Kimmich and J. Randles, Am. J. Physiol. 247 (Cell Physiol. 16): C74-C82, 1984]. The remaining models are terter ordered mechanisms with sodium debinding first at the trans side. If transfer across the membrane is rate limiting, the binding order can be determined to be sodium:sugar:sodium.