Stoichiometry versus coupling ratio in the cotransport of Na and different neutral amino acids

The stoichiometry of Na coupling to amino acid movement across the brush border membrane of the rabbit distal ileum has been determined under initial rate conditions. The coupling ratio, defined as the amino acid-dependent Na influx/the Na-dependent amino acid influx, was equal to unity for alanine, measured over a 10-fold range of Na and alanine concentrations. Coupling ratio values determined under a single set of conditions for a number of amino acids varied from 1 for serine to 4.6 for methionine. Reducing the methionine concentration from 12.5 to 1.5 mM caused the coupling ratio value to fall from 4.6 to 1.2. These results are explained by assuming a fixed stoichiometry of 1 : 1 under all conditions, with initial binding of the amino acids (A) to the Na-dependent carrier (E) but with some amino acids being able to cross on the Na-dependent carrier in the absence of Na. The variation in coupling ratio values can be used to calculate KA, the apparent dissociation constant of amino acid from the Na-dependent carrier in the absence of Na, and the ratio kappa 1/kappa 2, where kappa 1 and kappa 2 are first-order rate constants for translocation of the complexes EA and EANa, respectively. This method of processing results has been defined as delta analysis. The value of KA for methionine is 3.6 +/- 1.1 mM and the kappa 1/kappa 2 ratio is 1.01 +/- 0.07. The constant coupling ratio value for 1 for alanine indicates that the value for KA is extremely high or that the kappa 1 value is extremely low.