Nature of the cosubstrate action of Na+ and neutral amino acids in a transport system.

We have studied in the rabbit reticulocyte and the pigeon erythrocyte the interactions between Na+ and various neutral amino acids with respect to transport by an apparently ubiquitous Na+-dependent system that prefers the linear aliphatic and hydroxy aliphatic amino acids. In the series, alanine to norleucine, stronger and stronger apolar bonding to the transport site with increasing chain length is suggested. The interaction with Na+ is in contrast strong when the side chain is a methyl group, and weaker when it exceeds the length of the ethyl group. Other alkali metals will generally not serve. Instead they inhibit in competition with Na+. A hydroxyl group on carbon 3 or 4 of the amino acid leads to an even stronger interaction with Na+, but its effect becomes unf’avorable if it is farther removed from the carboxyl group. Mercapto and carboxamide groups produce similar effects. If the orientation of the hydroxyl group is tram with reference to the carboxylate group, a strong transport-producing interaction results; a cis orientation instead lowers the apparent affinity. On the basis of this and other evidence, we suggest that the two cosubstrates bind in juxtaposition at the receptor site, and that a substantial portion of the observed interaction occurs directly between them. This research provides the first evidence for the location of Na+ binding in a transport system specific for Na+ and a neutral substrate. The kinetics of this two-substrate system appears to be random order, with a difIerent step rate-limiting for transport of Na+ than of the amino acid.