FOLATE BINDERS IN HEMATOPOIESIS AND CELL REPLICATION

我们已经完成了明确确立这一比例的实验 肝细胞核有结合叶酸结合蛋白(FBP)的受体 从人慢性粒细胞白血病细胞中提纯。恰如其分 在实验中使用了对照组，以表明颗粒本身并不 把FBP绑在一起。标准的动力学研究和饱和度分析 使用FBP-[~3H]-叶酸络合物和125I-FBP与 核准备。进行了以下观察：(1)核 室温下结合FBP-[~3H]-叶酸络合物和~(125)I-FBP 4~C结合迅速，30min时接近最大值； 核上的结合部位是饱和的，通过Scatchard分析， 千道尔顿为0.7 nm的FBP-叶酸络合物，具有1,000个结合位点 (3)EDTA能抑制这种结合，这种抑制作用 被过量的钙离子逆转，但不被镁离子逆转。 用两种方法确定标记的FBP与 溶解的核：25%乙醇沉淀，凝胶过滤通过 Sephadex G-200。这两种方法都表明，从 使用Triton X-100的核将与FBP-叶酸复合体结合，这 结合被EDTA逆转。然而，问题是，收益率是 非常低，不足以进一步表征其特性。 为了从人体组织中纯化FBP，我们获得了脾(或 尸检或手术丢弃)来自骨髓增生性疾病患者 疾病。该组织中的FBP蛋白以两种形式存在：一种是小的 胞浆蛋白(约40千道尔顿)和一种膜蛋白 装订的(大约200千道尔顿)。我们现在正在提纯每一种蛋白质 来自组织匀浆的不同部分。 目前的目标是：(1)\纯化叶酸结合 人骨髓增殖性组织中的蛋白质及其性质的测定 和氨基酸组成；(2)溶解和表征核 与叶酸结合蛋白结合的受体；以及(3)决定 细胞内叶酸代谢的条件，如抗叶酸药物或 叶酸缺乏，它调节核受体的上或下 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)