N‐ethylmaleimide discriminates between two lysine transport systems in human erythrocytes.-猫眼课题宝

N-乙基马来酰亚胺区分人红细胞中的两个赖氨酸转运系统。

DOI：

发表时间：

1993

期刊：

Journal of Physiology

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通讯作者：

中科院分区：

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作者：

R. Devés;S. Angelo;P. Chávez

1.巯基试剂N-乙基马来酰亚胺（NEM）可抑制人红细胞中的低亲和力赖氨酸转运蛋白（系统y+），而不影响高亲和力转运蛋白（系统y+L）。2.用NEM预处理细胞可使L-[14 C]赖氨酸（1 μ M）的进入率降低约50%（最大效应）。3. NEM（0.2 mM）以单指数动力学抑制通量的NEM敏感组分。灭活速率常数（k，+/-S.E.M.）为0.53 +/-0.027 min-1（25 ℃）。底物不能防止失活。4.赖氨酸自抑制实验揭示了未处理细胞中的两种转运系统（半饱和常数Km; +/-S.E.M.），12.0+/-1.7 microM和109 +/-15.6 microM），并且在NEM处理的细胞中只有一个高亲和力系统（Km 9.5 +/-0.67 microM），表明NEM使系统y+失活。5. NEM-不敏感的L-[14 C]赖氨酸内流（系统y+L）被未标记的中性氨基酸以高亲和力抑制。L-亮氨酸在钠培养基中的抑制常数（Ki +/-S.E.M.）为10.7 +/-0.72 microM（37 ℃）。该系统也被L-甲硫氨酸、L-谷氨酰胺强烈抑制，而被L-苯丙氨酸和L-丝氨酸的亲和力较低。N-甲基-L-亮氨酸、L-脯氨酸和2-氨基-2-癸烷-羧酸（亮氨酸的双环类似物）没有发挥显著作用。6.在Na+、K+或Li+培养基中，赖氨酸通过y+L系统的转运以相同的速率发生，并且赖氨酸与转运蛋白的结合不受Na+置换的影响。7.系统y+L与中性氨基酸的相互作用依赖于介质中存在的阳离子。当Na+被K+取代时，亮氨酸和谷氨酰胺的抑制常数分别增加约90倍和60倍。Li+被证明是Na+的非常好的替代物。

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+.

Cloning of a rat kidney cDNA that stimulates dibasic and neutral amino acid transport and has sequence similarity to glucosidases.