We have found that erythroid burst promoting activity (BPA) is expressed by lymphocyte plasma membranes. We now propose to utilize a biochemically defined culture system to: 1. Purify membrane-associated BPA. We will utilize a variety of biochemical methods, including immunoaffinity, lectin affinity and gel filtration high-performance liquid chromatography (HPLC) and reverse-phase HPLC, to purify BPA from extracts of lymphocyte plasma membranes and shed vesicles. Purified fractions will be characterized by isoelectric focusing and sucrose gradient fractionation. Ultimately, purified BPA will be subjected to amino acid analysis. 2. Determine relatedness of soluble and vesicular BPA. We will employ antisera and monoclonal antibodies that react with BPA to detect whether purified BPA from membranes and in solution are antigenically related. Studies will be performed to determine if both physical forms of BPA are released by similar mechanisms from similar cell types, and if they act on similar marrow subpopulations. Target cells for each growth factor will be characterized histochemically and morphologically. 3. Define kinetics of vesicle-associated BPA release. Cumulative amounts and rates of vesiculation and BPA release by BPA-producing cell populations will be determined. Our findings will be correlated with studies aimed at measuring the biosynthesis of selected membrane components in culture. 4. Examine modes of BPA action. We will determine whether BPA shedding is a constitutive process or part of an immune reaction. Studies will be performed to examine whether vesiculation involves cytoskeletal components and whether membrane lipid and phospholipid composition is important to the shedding process. We will determine whether vesicles associate preferentially with marrow cells and, if so, what the nature of this association might be using electronmicroscopic methods. Ultimately, we will locate and purify BPA receptors with the projected long-range goal to examine intracellular mechanisms for receptor activation.
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