azaserine and 2-aminobicyclo(2-2-1)heptane-2-carboxylic-acid

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-[14C]Glutamine is transported in these membranes predominantly by Systems N/Nm (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 (less than 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'-methylhistidine were all strong inhibitors of glutamine uptake into liver membrane vesicles (greater than 65% inhibition at 20-fold excess), but neither homoglutamine nor N'-methylhistidine produced inhibition. L-Glutamate-gamma-hydroxamate was shown to be a competitive inhibitor of glutamine transport via System N (Ki 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.

Topics: Amino Acids; Amino Acids, Cyclic; Aminoisobutyric Acids; Animals; Azaserine; Biological Transport; Carrier Proteins; Cell Membrane; Diazooxonorleucine; Female; Glutamine; Kinetics; Lithium; Liver; Muscles; Rats; Rats, Inbred Strains; Sarcolemma; Sodium

Neutral amino acid uptake into mammalian cells occurs predominantly through the L, A, and ASC carrier-mediated transport systems. The proteins responsible for transport by these systems have not been isolated, and the three pathways presently are defined by their amino acid specificity and physiologic parameters. We have found that the amino acid derivative, O-diazoacetyl-L-serine (azaserine), is a potentially useful probe for identification of the L-(leucine-favoring) system transporter in human T-lymphocytes. Uptake of azaserine competitively inhibits the uptake of the prototype L-system amino acid, 2-amino-2-carboxybicycloheptane (BCH). Azaserine undergoes photolytic cleavage with 365 nm incident light to yield a highly reactive carbene intermediate and free N2. Following photolysis of [14C]azaserine in a suspension of lymphocytes, the 14C label is detectable within a crude cytoplasmic membrane preparation, and this process is inhibited by a 50-fold excess of unlabeled azaserine or 2-amino-2-carboxybicycloheptane, suggesting that the 14C-product is associated with the membranes at or near the L-system transport site. Furthermore, photolysis of azaserine in the presence of lymphocytes results in specific irreversible inhibition of L-system transport. Thus, photolysis of azaserine provides an initial step toward the identification of the L-system transporter.

Topics: Amino Acid Transport Systems; Amino Acids; Amino Acids, Cyclic; Azaserine; Biological Transport; Carrier Proteins; Humans; In Vitro Techniques; Kinetics; Light; Photolysis; T-Lymphocytes