Active alanine transport in isolated brush border membranes

Abstract

Uptake of L-alanine against a concentration gradient has been shown to occur with isolated brush border membranes from rat small intestine. An alanine transport system, displaying the following characteristics, was shown: (a) L-alanine was taken up and released faster than D-alanine; (b) Na+ as well as Li+ stimulated the uptake of both stereoisomers; (c) the uptake of L- and D-alanine showed saturation kinetics; (d) countertransport of L-alanine was shown; (e) other neutral amino acids inhibited L-alanine but not D-alanine entry when an electrochemical Na+ gradient across the membrane was present initially during incubation. No inhibition occurred in the absence of a Na+ gradient. The electrogenicity of L-alanine transport was established by three types of experiments: (a) Gradients of Na+ salts across the vesicle membrane (medium concentration greater than intravesicular concentration) supported a transient uptake of L-alanine above equilibrium level, and the lipophilic anion SCN- was the most effective counterion. (b) A gradient of K= across the membrane (vesicle greater than medium) likewise supported active transport of L-alanine into the vesicles provided the K= conductance of the membrane was increased with valinomycin. (c) Similarly, a proton gradient (vesicle greater than medium) in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an agent known to increase the proton conductance of membranes, produced an overshooting L-alanine uptake. A consideration of the possible forces, existing under the experimental conditions, suggests that the gradients of SCN-, K+ in the presence of valinomycin, and H+ in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone contribute to the driving force for L-alanine transport by creating a diffusion potential. Since the presence of Na+ was required in all experiments with active L-alanine transport these results support the existence of a transport system in the brush border membrane which catalyzes the co-transport of Na+ and L-alanine across this membrane.