MACROPHAGE DERIVED GROWTH FACTOR (MDGF)

Project Details

Description

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.
StatusFinished
Effective start/end date12/31/896/30/95

Funding

  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Cell Biology
  • Filtration and Separation

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