Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells.
Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells.
复制标题
人类白血病细胞叶酸结合蛋白的纯化、特性和免疫学特征。
DOI：
10.1016/0304-4165(87)90145-0
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Brink,L
中科院分区：
文献类型：
--
作者：
Sadasivan,E;daCosta,M;Rothenberg,SP;Brink,L
一种新的亲和层析基质，使用翼酰谷氨酸通过己二胺偶联到环氧活化基质，可以忽略配体泄漏，并允许从人脾脏中的人白血病细胞中纯化可溶性和膜相关叶酸结合蛋白。用2 M胍-盐酸从亲和基质中洗脱，从组织的溶解膜部分纯化了两种膜相关叶酸结合蛋白。高分子量结合蛋白的分子量约为31万，小分子量结合蛋白的分子量约为28万。通过sds -聚丙烯酰胺凝胶电泳，较小的膜相关蛋白的分子量为35 500，但由于样品中残留的高浓度Triton X-100干扰了凝胶的银染色，因此无法通过该方法测定较大的膜相关蛋白的分子量。通过sds -聚丙烯酰胺凝胶电泳，从44 000×gsupernatant组织匀浆液中通过酸洗脱纯化了两个叶酸结合蛋白，分子量分别为34 500和32 000。尽管从不同的细胞成分中纯化出可溶性和膜相关叶酸结合蛋白，但氨基酸组成是相似的，特别是极性氨基酸。所有这些形式的叶酸结合蛋白对氧化叶酸的亲和力高于对还原叶酸的亲和力，而对5-甲酰基四氢叶酸和甲氨蝶呤的亲和力非常低。尽管这些蛋白与先前从其他人类白血病细胞中提取的一种叶酸结合蛋白的抗血清发生交叉反应，但它们与从另外两种来源的人类白血病细胞、人类胎盘或人类KB细胞系中纯化的叶酸结合蛋白不发生交叉反应。
A new matrix for affinity chromatography using pteroylglutamic acid coupled to an epoxy-activated matrix via hexanediamine resulted in negligible ligand leakage and permitted the purification of soluble and membrane-associated folate-binding proteins from human leukemia cells contained in a human spleen. Two species of membrane-associated folate-binding proteins were purified from the solubilized membrane fraction of the tissue using 2 M guanidine-HCl to elute the proteins from the affinity matrix. The higher molecular weight binding protein had anMrof approximately 310 000 and the smaller species had anMrof approximately 28 000 by gel filtration. By SDS-polyacrylamide gel electrophoresis the smaller species of membrane-associated protein had a molecular weight of 35 500, but the molecular weight of the larger membrane-associated species could not be determined by this method because of the high concentration of residual Triton X-100 in the sample which interfered with the silver staining of the gel. Two folate-binding proteins, which by SDS-polyacrylamide gel electrophoreis had molecular weights of 34 500 and 32 000, were purified from the 44 000×gsupernatant fraction of the tissue homogenate by acid elution from the affinity matrix. Despite the different cell components from which the soluble and membrane-associated folate-binding proteins were purified, the amino acid compositions were similar, especially with respect to the apolar amino acids. All these forms of folate-binding proteins had higher affinity for oxidized than for reduced folates, and very low affinity for 5-formyltetrahydrofolate and methotrexate. Although these proteins cross-react with one antiserum raised previously to a folate-binding protein from other human leukemia cells, they do not cross-react with the folate-binding proteins purified from two other sources of human leukemia cells, from human placenta, or from the human KB cell line.