N-ethylmaleimide discriminates between two 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-[14C]lysine (1 microM) 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 Km; +/- S.E.M.), 12.0 +/- 1.7 microM and 109 +/- 15.6 microM) and only one high affinity system in NEM-treated cells (Km 9.5 +/- 0.67 microM), indicating that NEM inactivates system y+. 5. The NEM-insensitive L-[14C]lysine influx (system y+L) was inhibited with high affinity by unlabelled neutral amino acids. The inhibition constant for L-leucine in sodium medium (Ki +/- S.E.M.) was 10.7 +/- 0.72 microM (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+.
Devés
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R
Department of Physiology and Biophysics, Faculty of Medicine, University of Chile, Santiago.
Angelo
S
S
Chávez
P
P
D000596:Amino Acids / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine / Q000097:blood*; D011188:Potassium / Q000097:blood; D012964:Sodium / Q000097:blood
D000596:Amino Acids; D012964:Sodium; D008239:Lysine; D005033:Ethylmaleimide; D011188:Potassium