The isolation and properties of multiple forms of folate binding protein in cultured KB cells.
The isolation and properties of multiple forms of folate binding protein in cultured KB cells.
复制标题
培养的 KB 细胞中多种形式叶酸结合蛋白的分离和特性。
DOI：
10.1016/0003-9861(86)90705-8
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发表时间：
1986
影响因子：
3.9
通讯作者：
Rothenberg,SP
中科院分区：
文献类型：
--
作者：
Luhrs,CA;Sadasivan,E;daCosta,M;Rothenberg,SP
在正常 (N) 和叶酸缺乏 (D) 培养基中培养的 KB 细胞的叶酸结合蛋白 (FBP) 已被表征。 200,000g裂解细胞上清液含有两个FBP，可以通过DEAE-Bio-Gel A色谱分离，表明它们的离子电荷不同，尽管它们不能通过Sephadex G-100凝胶过滤分离（表观Mr~40,000）。在用 Triton X-100 溶解后，通过 Sephadex G-150 进行凝胶过滤，在细胞膜成分中鉴定出两种 FBP，即表观 Mr~160,000 的主要形式和表观 Mr~40,000 的次要形式。通过亲和层析从培养这些细胞的培养基中分离和纯化额外的FBP。通过凝胶过滤和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，该FBP的表观Mr约为44,000。 200,000g 细胞裂解物上清液、培养基和 Triton 溶解的膜中 FBP 的蝶酰谷氨酸的缔合常数相似，并且叶酸类似物与 FBP 的相对亲和力（相对于蝶酰谷氨酸）对于所有物种都是相似的。针对从培养基中纯化的FBP而产生的抗血清沉淀了200,000g细胞裂解物上清液、Triton溶解的膜和培养基中的FBP，表明这些FBP之间具有抗原同源性。当KB细胞在N培养基中培养时，200,000g细胞裂解物上清液或培养基中没有不饱和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的不饱和 FBP 相当大（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（4.5 至 756 pmol/mg 蛋白质）中培养时，不饱和 FBP 也会增加，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的总叶酸结合能力是正常KB细胞的两倍，表明FBP的诱导。
The folate binding proteins (FBPs) of KB cells which were cultured in normal (N) and folate-deficient (D) medium have been characterized. The 200,000gsupernate 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~40,000). Two species of FBP, a major form of apparent Mr~160,000 and a minor form of apparentMr~ 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 apparentMrof this FBP was ~ 44,000. The association constants for pteroylglutamic acid of the FBPs in the 200,000gcell 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 200,000gcell lysate supernate, Triton-solubilized membrane, and culture medium, indicating antigenic homology among these FBPs. There was no unsaturated FBP in the 200,000gcell 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 200,000gcell 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.