SUBSTRATE-SPECIFICITY OF GLUTAMINE TRANSPORTERS IN MEMBRANE-VESICLES FROM RAT-LIVER AND SKELETAL-MUSCLE INVESTIGATED USING AMINO-ACID-ANALOGS

We investigated the effects of glutamine and histidine analogues on glutamine transport processes in membrane vesicles prepared from rat liver (sinusoidal membrane) and skeletal muscle (sarcolemma). L-[C-14]Glutamine is transported in these membranes predominantly by Systems N/N(m) (liver and muscle respectively), and to a lesser extent by Systems A and L (e.g. about 60, 20 and 20% of total flux respectively via Systems N, A and L at 0.05 mM-glutamine in liver membrane vesicles). The glutamine anti-metabolites 6-diazo-5-oxo-L-norleucine and acivicin were relatively poor inhibitors of glutamine uptake into liver membrane vesicles (< 25% inhibition at 20-fold excess) and appeared primarily to inhibit System A activity (i.e. N-methylaminoisobutyric acid-inhibitable glutamine uptake). In similar experiments azaserine (also a glutamine anti-metabolite) inhibited approx. 50 % of glutamine uptake, apparently by inhibition of System A and also of System L (i.e. 2-amino-2-carboxybicyclo[2,2,1]heptane-inhibitable glutamine uptake). Glutamate gamma-hydroxamate, aspartate beta-hydroxamate, histidine and N-pi-methylhistidine were all strong inhibitors of glutamine uptake into liver membrane vesicles (> 65% inhibition at 20-fold excess), but neither homoglutamine nor N-tau-methylhistidine produced inhibition. L-Glutamate-gamma-hydroxamate was shown to be a competitive inhibitor of glutamine transport via System N (K(i) approximately 0.6 mM). Glutamine uptake in sarcolemmal vesicles showed a similar general pattern of inhibition as in liver membrane vesicles. The results highlight limits on the substrate tolerance of System N; we suggest that the presence of both an L-alpha-amino acid group and a nitrogen group with a delocalized lone-pair of electrons (amide or pyrrole type), separated by a specific intramolecular distance (C2-C4 chain equivalent), is important for substrate recognition by this transporter.