Structural and functional analysis of a cystine/glutamate-specific carrier protein of plasma membrane

应激剂诱导体外培养的小鼠巨噬细胞的半胱氨酸转运活性。诱导半胱氨酸进入细胞可以增强谷胱甘肽的合成，谷胱甘肽是一种抗氧化的细胞物质。我们尝试通过差异筛选的方法克隆半胱氨酸载体，以筛选胁迫诱导克隆。我们从1.5-3kb的小鼠巨噬细胞mRNA1.5-3kb片段中构建了定向cDNA文库，从约8×10~(-4)噬菌体斑块中挑选了800个克隆。虽然我们没有分离到编码胱氨酸载体的CNDA克隆，但我们成功地克隆了一个应激诱导的23 kDa蛋白MSP23。该蛋白属于一种新型的抗氧化蛋白。此外，我们还发现，另外两种已知蛋白质在暴露于氧化应激时也会被诱导。我们还获得了几个编码新的应激诱导蛋白的cDNA克隆。

Stress agents induces cystine transport activity in mouse macrophages cultured in vitro. The induction of cystine entry into the cells enhances synthesis of glutathione, an anti-oxidative cellular substance. We tried to clone the carrier of cystine by the differential screening to select stress-induced clones. We constructed a directional cDNA library from 1.5-3 kb fraction of mouse macrophage mRNA.We selected total 800 clones from about 8x10^4 phage plaques. We could not detect any expression of the cystine transport activity in Xenopus laevis oocytes after microinjection of cRNAs synthesized from those cDNAs.Although we could not isolate a cNDA clone encodnig cystine carrier, we have succeeded in the cloning of a stress-induced 23 kDa protein named MSP23. This protein belongs to a new type of antioxidative proteins. Additionally we found that two other known proteins were induced upon exposure to oxidative stress. We also obtained a few cDNA clones that encode novel stress-induced proteins.