N-ETHYLMALEIMIDE DISCRIMINATES BETWEEN 2 LYSINE TRANSPORT-SYSTEMS IN HUMAN ERYTHROCYTES

1. The sulfhydryl reagent N-ethylmaleimide (NEM) was shown to inactivate the low affinity lysine transporter in human erythrocytes (system y+) without affecting the high affinity transporter (system y+L). 2. Pre-treatment of the cells with NEM reduced the rate of entry Of L-[C-14]lysine (1 muM) by approximately 50 % (maximum effect). 3. NEM (0.2 mm) inhibited the NEM-sensitive component of the flux with mono-exponential kinetics. The inactivation rate constant (k, +/- S.E.M.) was 0.53 +/- 0-027 min-1 (25-degrees-C). The substrate did not protect against inactivation. 4. Lysine self-inhibition experiments revealed two transport systems in untreated cells (half-saturation constants K(m); +/- S.E.M.), 12.0 + 1.7 muM and 109 +/- 15.6 muM) and only one high affinity system in NEM-treated cells (K(m) 9-5 +/- 0-67 muM), indicating that NEM inactivates system y+. 5. The NEM-insenSitiVe L-[C-14]lysine influx (system y+L) was inhibited with high affinity by unlabelled neutral amino acids. The inhibition constant for L-leucine in sodium medium (K(i) +/- S.E.M.) was 10-7 +/- 0-72 muM (37-degrees-C). The system was also strongly inhibited by L-methionine, L-glutamine and with less affinity by L-phenylalanine and L-serine. N-methyl-L-leucine, L-proline and 2-amino-2-norbornane-carboxylic acid, a bicyclic analogue of leucine, did not exert a significant effect. 6. Lysine transport through system y+L occurred at the same rate in Na+, K+ or Li+ medium and the binding of lysine to the transporter was unaffected by Na+ replacement. 7. The interaction of system y+L with neutral amino acids was dependent on the cation present in the medium. The inhibition constant for leucine and glutamine increased approximately 90- and 60-fold respectively when Na+ was replaced by K+. Li+ was shown to be a very good substitute for Na+.