SUMMARY1. 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 ftM) 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-' (25°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 /tM and 109 + 156 jsM) and only one high affinity system in NEM-treated cells (Km 9-5 + 0-67 /IM), indicating that NEM inactivates system y+.5. The NEM-insensitive L-[j4C]lysine influx (system y+L) was inhibited with high affinity by unlabelled neutral amino acids. The inhibition constant for L-leucine in sodium medium (K1 ± S.E.M.) was 10-7 + 0-72 ,lM (37°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+.
The transport specificity of system y+L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of L-[14C]-lysine. Some neutral amino acids, L-lysine and L-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external L-leucine and L-lysine increased the rate by 5.3 +/- 0.63 and 6.2 +/- 0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (L-tryptophan, L-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (L-alanine, L-serine). Half-saturation constants (+/- SEM) calculated from this effect (L-lysine, 10.32 +/- 0.49 microM and L-leucine, 11.50 +/- 0.50 microM) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na+, Li+ or K+ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na+ or Li+ were replaced by K+. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.
The sucrose-induced stimulation of lysine influx in human erythrocytes has been attributed to the removal of a competitive inhibition exerted by Na ؉ on system y . NaCl replacement with sucrose increased influx through system y ؉ L, but decreased influx through system y ؉ . We conclude that 1) the inhibition of system y ؉ is a response to the membrane depolarization that results from chloride removal, and 2) the stimulation of system y ؉ L is due to the enhancement of the negative surface potential. Consistently, lysine influx through system y ؉ L (in sucrose medium) was reduced by Na Several transport systems for cationic permeants have been proposed to contain an Na ϩ binding region within the substrate site. These include the cationic amino acid transporter (system y ϩ ) in human erythrocytes (1), the broad-scope amino acid transporter (b 0,ϩ ), and the choline transporter in mouse blastocysts (2, 3). This hypothesis was postulated to explain the increase in the rate of entry that occurs when the standard NaCl medium is replaced by isotonic sucrose and the reduction in the apparent K m for the cationic substrate that results from the same substitution. In erythrocytes, lysine uptake (5 M, 20°C) was found to be 3-fold higher in isotonic sucrose medium than in 150 mM NaCl, while the apparent K m changed from 59 M in NaCl to 15 M in sucrose (1). Similarly, in blastocysts, Na ϩ replacement by uncharged osmolites induced an approximately 3-fold increase in lysine influx (0.42 M, 37°C), which was accompanied by a shift in apparent K m from 61 to 5 M (2).The hypothesis stating that an Na ϩ binding site exists within the substrate site of cationic amino acid transporters has given rise to some considerations of general significance. It has been remarked that it is consistent with the proposal that the Na ϩ
SUMMARYThe properties are discussed of system y+L, a broad scope amino acid transporter which was first identified in human erythrocytes. System y+L exhibits two distinctive properties: (a) it can bind and translocate cationic and neutral amino acids, and (b) its specificity varies depending on the ionic composition of the medium. In Na+ medium, the half-saturation constant for L-lysine influx was 9.5 ± 0.67 /LM and the inhibition constant (K1) for L-leucine was 10.7 ± 0.72 ,EM.L-Leucine is the neutral amino acid that binds more powerfully, whereas smaller analogues, such as L-alanine and L-serine interact less strongly (the corresponding inhibition constants were K1,Alas 0.62 ± 0.11 mM; Ki,Serv 0-49 + 0.08 mM). In the presence of K+ the carrier functions as a cationic amino acid specific carrier, but Li+ is able to substitute for Na+ facilitating neutral amino acid binding. The effect of the inorganic cations is restricted to the recognition of neutral amino acids; translocation occurs at similar rates in the presence of Na+, K+ and Li+.The only structural feature that appears to impair translocation is bulkiness and substrates with half-saturation constants differing by more than 100-fold translocate at the same rate. This suggests that translocation is largely independent of the forces of interaction between the substrate and the carrier site. System y+L activity has been observed in Xenopus laevis oocytes injected with the cRNA for the heavy chain of the 4F2 human surface antigen. 4F2hc is an integral membrane protein with a single putative membrane-spanning domain and it remains to be clarified whether it is part of the transporter or an activator protein.
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