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

1. The sulfhydryl reagent N-ethylmaleimide (NEM) was shown to inactiva te the low affinity lysine transporter in human erythrocytes (system y +) without affecting the high affinity transporter (system y+L). 2. Pr e-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-exponent ial 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 trans port 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 affinit y 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 infl ux (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 aff inity by L-phenylalanine and L-serine. N-methyl-L-leucine, L-proline a nd 2-amino-2-norbornane-carboxylic acid, a bicyclic analogue of leucin e, did not exert a significant effect. 6. Lysine transport through sys tem y+L occurred at the same rate in Na+, K+ or Li+ medium and the bin ding 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 leuc ine and glutamine increased approximately 90- and 60-fold respectively when Na+ was replaced by K+. Li+ was shown to be a very good substitu te for Na+.