FOLATE BINDERS IN HEMATOPOIESIS AND CELL REPLICATION

我们完成的实验明确地证实了那只老鼠

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)