Fan J, Vitols K S, Huennekens F M
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
Adv Enzyme Regul. 1992;32:3-15. doi: 10.1016/0065-2571(92)90005-k.
Biotin derivatives of methotrexate (biotin-SS-MTX) and folate (biotin-SS-folate), in which the functional components are joined by a dissociable disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by HPLC, elemental analysis and mass spectrometry. These compounds provide a convenient means for the single-step purification of the folate transporters from L1210 cells. Parental L1210 murine leukemia cells, which contain only the microM transporter (the reduced folate/MTX transport protein) were treated with the N-hydroxysulfosuccinimide ester of biotin-SS-MTX, and a detergent extract of the plasma membranes was exposed to streptavidin-agarose beads to adsorb the labeled protein. Dithiothreitol cleavage of the disulfide linkage released the transporter, which migrated as a well-defined component (43 kDa) on SDS-PAGE gels; no other proteins were present. An L1210 subline (JF), obtained by adapting cells to grow on nanomolar concentrations of folate, contains both the microM transporter and the nM transporter (high-affinity folate binding protein). When these cells were treated with the N-hydroxysulfosuccimide ester of biotin-SS-folate and processed as described above, analysis on SDS-PAGE gels revealed the presence of two proteins, the microM transporter (43 kDa) and the nM transporter (39 kDa). Both transporters were characterized with respect to amino acid content; blocked N-termini precluded Edman sequencing. Treatment of the nM transporter with peptide:N-glycosidase F produced a smaller component (32 kDa); the microM transporter, conversely, was unchanged by this procedure. When the microM transporter in parental L1210 cells was labeled with fluorescein-MTX and then treated with phosphoinositol-specific phospholipase C (PI-PLC), no change in fluorescence was detected. Alternatively, when the nM transporter in the JF subline was labeled with fluorescein-folate and then treated with PI-PLC, complete loss of fluorescence was observed. These results indicate that the L1210 microM transporter is a non-glycosylated, integral membrane protein, while its nM counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.
已合成了甲氨蝶呤（生物素-SS-甲氨蝶呤）和叶酸（生物素-SS-叶酸）的生物素衍生物，其中功能成分通过可解离的含二硫键间隔基连接，经DEAE-三羟甲基氨基甲烷琼脂糖色谱法纯化，并通过高效液相色谱、元素分析和质谱进行表征。这些化合物为从L1210细胞中一步纯化叶酸转运蛋白提供了便利方法。仅含有微摩尔转运蛋白（还原型叶酸/甲氨蝶呤转运蛋白）的亲本L1210小鼠白血病细胞用生物素-SS-甲氨蝶呤的N-羟基琥珀酰亚胺酯处理，质膜的去污剂提取物与链霉亲和素琼脂糖珠接触以吸附标记的蛋白质。二硫键的二硫苏糖醇裂解释放出转运蛋白，其在SDS-PAGE凝胶上迁移为一个明确的组分（43 kDa）；不存在其他蛋白质。通过使细胞适应在纳摩尔浓度的叶酸上生长而获得的L1210亚系（JF）同时含有微摩尔转运蛋白和纳摩尔转运蛋白（高亲和力叶酸结合蛋白）。当这些细胞用生物素-SS-叶酸的N-羟基琥珀酰亚胺酯处理并按上述方法处理时，SDS-PAGE凝胶分析显示存在两种蛋白质，即微摩尔转运蛋白（43 kDa）和纳摩尔转运蛋白（39 kDa）。对两种转运蛋白的氨基酸含量进行了表征；封闭的N末端排除了埃德曼测序。用肽：N-糖苷酶F处理纳摩尔转运蛋白产生一个较小的组分（32 kDa）；相反，微摩尔转运蛋白在此过程中未发生变化。当亲本L1210细胞中的微摩尔转运蛋白用荧光素-甲氨蝶呤标记，然后用磷脂酰肌醇特异性磷脂酶C（PI-PLC）处理时，未检测到荧光变化。或者，当JF亚系中的纳摩尔转运蛋白用荧光素-叶酸标记，然后用PI-PLC处理时，观察到荧光完全丧失。这些结果表明，L1210微摩尔转运蛋白是一种非糖基化的整合膜蛋白，而其对应的纳摩尔转运蛋白则高度糖基化，并通过糖基磷脂酰肌醇组分从膜外锚定在膜上。
