Greene A A, Marcusson E G, Morell G P, Schneider J A
Department of Pediatrics, University of California, San Diego, La Jolla 92093.
J Biol Chem. 1990 Jun 15;265(17):9888-95.
We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.
我们在此对小鼠L-929成纤维细胞中的一种溶酶体胱氨酸转运体进行了表征。用胱氨酸预加载的细胞的颗粒组分表现出反向转运，且该转运不依赖于Na⁺或K⁺。无限转运流入的米氏常数为0.27±0.06 mM（n = 3），并且观察到胱氨酸进入存在一个非饱和成分，其解离常数Kd为0.8 - 1.8 nmol胱氨酸·min⁻¹·己糖胺酶-1单位·mM⁻¹。我们没有发现证据表明胱氨酸也通过任何其他已知的溶酶体氨基酸转运体进行转运。测试了50多种类似物抑制反向转运的能力。报告了硒代胱氨酸、胱硫醚、硒代蛋氨酸和亮氨酸的抑制常数。该转运体识别的重要要求是存在氨基、L构型以及链长不超过八个原子。不需要净正电荷或负电荷。一些二极和四极氨基酸也能被识别。我们推测结合位点具有极性和非极性结构域，后者足够大以容纳C-3位的分支以及硒或碳取代硫的情况。
