The specificity of the neutral amino acid transport system in the brush border was examined by studying the ability of amino acid analogues to inhibit the unidirectional influx of phenylalanine from mucosal solution into the cells. Effects were evaluated in terms of the affinity of various substrates for the amino acid site in the transport system. The affinity of amino acids for the site was proportional to the number of carbon atoms in the side chain. Electronwithdrawing substituents in the ring of phenylalanine increased affinity and electron-releasing groups decreased affinity. Removal of the a-amino group from phenylalanine decreased affinity by a factor of approximately 50 and removal of the carboxyl group decreased affinity 12-fold. Effects on affinity of variations in the side chain of the amino acid can be comparable in magnitude to that of the carboxyl group. The effect of sodium ion on the transport system appears to be similar for all compounds tested.The transport of neutral (monoamino-monocarboxylic) amino acids by rabbit ileum has been studied extensively in this laboratory. We have characterized the influx of several amino acids across the brush border and from this information have developed a model of the transport system located in this membrane (1). This model provides a satisfactory explanation of the interaction between Na and amino acids for influx from mucosal solution into the cell (1-4) and for efflux from the cell toward the mucosal solution (5, 6). The model involves sequential binding of the amino acid and Na to a transport site, and the behavior of the system can be characterized by dissociation constants for the reaction of amino acid with the site and the reaction of Na with the site. Since this model fits data for the influxes of glycine, alanine, valine, and leucine, we felt that it would be worthwhile to attempt to obtain further information about the nature of the transport site by investigating the behavior of a variety of amino acids and their analogues. There have been previous studies of this type that have led to certain general conclusions regarding the 673
The model of the interaction between Na and alanine at the mucosal border of rabbit ileum has been tested further by examining the efflux of alanine from the cells toward the mucosal solution. Alanine efflux shows a tendency toward saturation as cellular alanine concentration increases and is influenced by cellular Na concentration. A decrease in cell Na concentration causes an increase in the apparent Michaelis constant with little change in maximal efflux rate. Studies on strips of mucosa treated with ouabain or cyanide showed that the direction of net alanine transfer between the cells and the medium is determined by the direction of the Na concentration difference. The cells extrude alanine against a concentration difference when cell [Na] exceeds medium [Na] and accumulate alanine when cell [Na] is less than medium [Na]. The observations are consistent with the model previously suggested involving a transport site that combines with and translocates both Na and alanine, and with the concept that the Na concentration difference between mucosal solution and cytoplasm provides at least part of the energy for active transport of alanine.In a series of earlier studies, certain aspects of the interaction between Na and alanine transport in rabbit ileum (1-4) were examined in some detail. Those studies dealt mainly with the unidirectional influxes of Na and alanine across the brush border from the mucosal solution into the cells. They led to a model of the transport system involving a site that is capable of combining with both Na and alanine, and that can result in the translocation of both solutes across the brush border membrane. The influx of alanine via this mechanism is not markedly affected by metabolic inhibitors (4), suggesting that a direct link between metabolic energy and the transport system may not be involved. Further, alanine influx was not appreciably altered by ouabain, suggesting that the Na dependence of the process is not mediated by a Na-sensitive ATPase that is inhibited by ouabain. We felt that these ob-277
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