FOLATE BINDERS IN HEMATOPOIESIS AND CELL REPLICATION

我们已经完成了实验，明确地确定， 肝细胞核具有结合叶酸结合蛋白（FBP）的受体 从人慢性髓性白血病细胞中纯化。 适当 在实验中使用对照以表明单独的颗粒不 绑定FBP。 标准动力学研究和饱和度分析如下： 使用FBP-[3H]-叶酸复合物和 125I-FBP与 核准备 观察到：（1）细胞核 绑定两个FBP-[3H]-叶酸复合物和 125室温下I-FBP 和4~C. 结合迅速，30 min时接近最大值;（2） 核上的结合位点是饱和的，通过Scatchard分析， 0.7 nM的FBP-叶酸复合物的千道尔顿，具有1，000个结合位点 （3）EDTA可抑制这种结合， 被过量的钙逆转2个以上 但不是Mg2个以上. 使用两种方法来确定标记的FBP与标记的MBP的结合。 溶解的核：在25%乙醇中沉淀并通过凝胶过滤 Sephadex G-200。 这两种方法都表明， 使用Triton X-100的细胞核将结合FBP-叶酸复合物， 结合被EDTA逆转。 然而，问题是， 非常低，不足以进一步表征其性质。 为了从人组织中纯化FBP，我们获得了脾（或者 尸检或手术丢弃物） 疾病 该组织中的FBP蛋白以两种形式存在： 胞质溶胶蛋白（约40千道尔顿）和一种是膜蛋白 束缚态（约200千道尔顿）。我们现在正在纯化每一种蛋白质 来自组织匀浆的不同部分。 目前的目标是：（1）将叶酸结合蛋白纯化至均一， 从人骨髓增生组织中提取蛋白质并测定其性质 和氨基酸组成;（2）溶解和表征核 结合叶酸结合蛋白的受体;和（3）\确定 细胞内叶酸代谢的条件，如抗叶酸药物或 叶酸缺乏症，它调节向上或向下的核受体， 叶酸结合蛋白 （B）

We have completed experiments which unambiguously established that rat liver nuclei have receptors which bind the folate-binding protein (FBP) purified from human chronic myelogenous leukemia cells. Appropriate controls were used in the experiments to show that granules alone did not bind the FBP. Standard kinetic studies and saturation analyses were carried out using the FBP-[3H]-folate complex and 125I-FBP with the nuclear preparation. The following observations were made: (1)\the nuclei bind both the FBP-[3H]-folate complex and 125I-FBP at room temperature and 4~C. Binding is rapid, reaching near maximal values by 30 min; (2)\the binding sites on the nuclei are saturable and by Scatchard analysis had a kilodalton for the FBP-folate complex of 0.7 nM, with 1,000 binding sites per nucleus; and (3)\the binding is inhibited by EDTA and this inhibition is reversed by excess Ca2+ but not by Mg2+. Two methods were used to ascertain the binding of the labeled FBP by the solubilized nuclei: precipitation at 25% ethanol and gel-filtration through Sephadex G-200. Both methods show that a macromolecule solubilized from the nuclei using Triton X-100 will bind the FBP-folate complex and this binding is reversed by EDTA. The problem, however, is that the yield is very low and insufficient for further characterization of its properties. For purification of the FBP from human tissues, we obtained spleens (either autopsy or surgical discards) from patients with myeloproliferative diseases. The FBP protein in this tissue exists in two forms: a small cytosol protein (approximately 40 kilodaltons) and one which is membrane bound (approximately 200 kilodaltons). We are now purifying each protein from different fractions of the tissue homogenate. Present objectives are: (1)\purify to homogeneity the folate-binding protein from human myeloproliferative tissue and determine its properties and amino acid composition; (2)\solubilize and characterize the nuclear receptor which binds the folate-binding protein; and (3)\determine the conditions of intracellular folate metabolism, such as antifolate drugs or folate deficiency, which modulate up or down the nuclear receptor for the folate-binding protein. (B)