Luhrs C A, Sadasivan E, da Costa M, Rothenberg S P
Arch Biochem Biophys. 1986 Oct;250(1):94-105. doi: 10.1016/0003-9861(86)90705-8.
The folate binding proteins (FBPs) of KB cells which were cultured in normal (N) and folate-deficient (D) medium have been characterized. The 200,000 g supernate of lysed cells contained two FBPs which could be separated by DEAE-Bio-Gel A chromatography, indicating that they differ in ionic charge although they could not be separated by gel filtration through Sephadex G-100 (apparent Mr approximately 40,000). Two species of FBP, a major form of apparent Mr approximately 160,000 and a minor form of apparent Mr approximately 40,000, were identified by gel filtration through Sephadex G-150 in the membrane component of the cells after solubilization with Triton X-100. An additional FBP was isolated and purified by affinity chromatography from the medium in which these cells were cultured. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent Mr of this FBP was approximately 44,000. The association constants for pteroylglutamic acid of the FBPs in the 200,000 g cell lysate supernate, culture medium, and Triton-solubilized membrane were similar and the relative affinity of folate analogs for the FBP, vis-à-vis pteroylglutamic acid, was similar for all species. An antiserum raised to the purified FBP from the culture medium precipitated the FBPs in the 200,000 g cell lysate supernate, Triton-solubilized membrane, and culture medium, indicating antigenic homology among these FBPs. There was no unsaturated FBP in the 200,000 g cell lysate supernate or medium when KB cells were cultured in N medium. However, when cells were cultured in D medium, the unsaturated FBP of the 200,000 g cell supernate and culture medium was substantial (9.2 and 14.1 pmol/mg protein, respectively). Unsaturated FBP was detected in the membrane of normal cells but this also increased when these cells were cultured in D medium (4.5 to 756 pmol/mg protein), indicating that the FBPs of these cellular compartments are normally saturated by folate. After 16 weeks of culture in D medium, the total folate binding capacity of the membrane-associated FBP was twofold greater than that of normal KB cells, indicating the induction of FBP.
对在正常（N）培养基和叶酸缺乏（D）培养基中培养的KB细胞的叶酸结合蛋白（FBP）进行了特性分析。裂解细胞的200,000g上清液中含有两种FBP，它们可通过DEAE - Bio - Gel A色谱法分离，这表明它们在离子电荷上存在差异，尽管通过Sephadex G - 100凝胶过滤无法分离（表观分子量约为40,000）。在用Triton X - 100溶解细胞后，通过Sephadex G - 150凝胶过滤在细胞的膜组分中鉴定出两种FBP，一种主要形式的表观分子量约为160,000，一种次要形式的表观分子量约为40,000。通过亲和色谱法从这些细胞培养的培养基中分离并纯化了一种额外的FBP。通过凝胶过滤和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，这种FBP的表观分子量约为44,000。200,000g细胞裂解液上清液、培养基和Triton溶解的膜中FBP对蝶酰谷氨酸的结合常数相似，并且叶酸类似物相对于蝶酰谷氨酸对FBP的相对亲和力在所有种类中也相似。针对从培养基中纯化的FBP产生的抗血清沉淀了200,000g细胞裂解液上清液、Triton溶解的膜和培养基中的FBP，表明这些FBP之间存在抗原同源性。当KB细胞在N培养基中培养时，200,000g细胞裂解液上清液或培养基中不存在不饱和FBP。然而，当细胞在D培养基中培养时，200,000g细胞上清液和培养基中的不饱和FBP含量可观（分别为9.2和14.1 pmol/mg蛋白质）。在正常细胞的膜中检测到不饱和FBP，但当这些细胞在D培养基中培养时其含量也增加（从4.5至756 pmol/mg蛋白质），这表明这些细胞区室的FBP通常被叶酸饱和。在D培养基中培养16周后，膜相关FBP的总叶酸结合能力比正常KB细胞高两倍，表明FBP被诱导产生。
