Further Studies of Amino Acid Transport by Embryonic Chick Bone

Uptakes by embryonic chick bone of various neutral amino acids and the effects on these uptakes of low temperature, N -ethylmaleimide (NEM), and competing amino acids were measured. At 38°, diffusible radioactivity gradients of more than 1.0 between total tissue water and incubation medium were established for each of 15 amino acids tested. At 4°, by contrast, entry of amino acids into the intracellular fraction was slow and did not proceed against a concentration gradient. Amino acid accumulation was completely inhibited by NEM. This inhibition was not immediate, however, and, during the first ½ hour of exposure to NEM, amino acid gradients were developed. Thereafter, net efflux occurred until, in some experiments, virtually all of the free amino acid had been expelled from the cells. If the bones in the presence of NEM were incubated at 4° after the first 30 min at 38°, net efflux did not occur; instead, the 30-min gradients were retained unchanged for at least 2½ hours. It was concluded that the NEM-treated tissue is capable of driving amino acid efflux by a metabolically dependent process despite inhibition of influx capacity. In this respect, inhibition by NEM differs from inhibition by actinomycin D, puromycin, or chilling. Evidence is presented for the existence of at least two sites for the transport of neutral amino acids by embryonic bones. The sites appear to be analogous to the alanine-preferring ( A ) and leucine-preferring ( L ) sites described by Christensen and colleagues. The A site specificity, as indicated by sensitivity to inhibition by alanine, is especially high for proline and α-aminoisobutyric acid. l site dependence is most notable for isoleucine, and in this tissue is defined more clearly by sensitivity to tryptophan than by sensitivity to leucine. Leucine has mixed A and L site affinities, as do most of the other amino acids tested.