Macrophages secrete an activity termed "macrophage-derived growth factor", MDGF, that stimulates the proliferaction of several mesenchymal cell types. Macrophage cell lines (P-388DI and J-774-2) produce MDGF in culture. We have achieved and 8500 fold purification of MDGF from both rat peritoneal macrophages and P-388DI and J-774-2 conditioned media, and have characterised several properties of this substance. This proposal is designed to: (a) Prepare large scale cultures of P-388DI cells, for the preparation of conditioned media for the purification of MDGF. (b) Purify MDGF to homogeneity. Ammonium sulfate precipitation, DEAE-Biogel ion exhange chromatography, Sephacryl S-200 chromatography and iso-electric focusing will be utilized, based on the purification we have achieved to date. High performancve liquid chromatography (HPLC), including reversed phase HPLC, will be utilized as additional purification steps. (c) Raise specific antibodies to MDGF in rabbits and to purify them using affinity columns. (d) Prepare anti-MDGF antiboty afinity columns, for potential purification of MDGF. (e) Prepare a high specific activity, biologically active radiolabelled (125I) derivative of MDGF, for use in binding studies. (f) Develoop a radioimmunoassay (RIA) for measuring MDGF. (g) Develop an enzyme-linked immunoadsorbent assay (ELISA) for detection of specific anti-MDGF antibodies. (h) Identify and characterize cellular receptors for MDGF on fibroblasts. Specificity, reversibility and saturability of binding, and the molecularity and the equilibrium association constant of binding of MDGF to its putative receptor will be determined. The relationship of receptor occupancy to biological activity will be examined. (i) Study cellular uptake of MDGF, receptor "down-regulation", and the requirement for intracellular processing for biological activity. (j) Characterize the MDGF receptor physicohemically, by using cleavable crosslinking reagents to covalently link MDGF to its receptor in the cell membrane. (k) Biochemically characterize purified MDGF. Amino acid and polypeptide chein compositions will be determined, and peptide mapping (trypsin, chymotrypsin and cyanogen bromide) will be carried out. These studies should help elucidate the nature, properties and mode of action of MDGF.
NIH ExPorter
NIH ExPorter