Hemopoietic growth factor purification and action

Abstract

Hemopoiesis involves a continuous process of cell proliferation and differentiation which leads to the production of functional circulatory blood cells. The proliferation and maturation of hemopoietic cells is dependent on specific regulatory molecules known as hemopoietin growth factors (HGFs). A multilineage HGF, hemopoietin 1 (H-1), was purified from the conditioned medium of the 5637 human bladder carcinoma cell line. The HGF activity of the conditioned medium resulted from the activities of two charged species pI = 4.8, {dollar}\sim{dollar}85%, pI = 5.3, {dollar}\sim{dollar}15%. A four step procedure consisting of ion exchange chromatography, chromatofocusing, gel filtration and hydrophobic chromatography, was developed for the more abundant species. Purified H-1 possessed a molecular weight of {dollar}\sim{dollar}17,000 on reduced SDS-PAGE and a molecular weight of {dollar}\sim{dollar}18,000 of SDS-PAGE under non-reducing conditions. Both radiolabeled reduced and non-reduced H-1 behaved as a single homogeneous band which co-migrated with the biological activity on polyacrylamide gels. Initial studies indicate that H-1 acts on the most primative hemopoietic cells yet shown to proliferate in vitro.;In a separate study, protein phosphorylation was investigated as a mechanism by which the mononuclear phagocyte HGF, CSF-1 mediates its biological response. CSF-1 was shown to stimulate the phosphorylation of proteins in mouse macrophage membrane preparations. CSF-1 induced phosphorylation was specific, saturable, and concentration dependent. The number of proteins as well as the amount of phosphate incorporated was dependent on the ATP concentration, the divalent cations present as well as the cell type.;Membranes prepared from cells of a CSF-1 receptor bearing cell line with decreased growth responsiveness to CSF-1 exhibited restricted phosphorylation patterns. In all 3 macrophage cell types examined CSF-1 increased phosphate incorporation into at least 3 proteins. One of these proteins was the CSF-1 receptor, which when purified exhibited CSF-1 stimulated auto-phosphorylation in tyrosine residues. In the absence of divalent cations, CSF-1 stimulated phosphorylation of membrane protein occurred without phosphorylation of the receptor. Other effects of CSF-1 included an inhibition in the phosphorylation of a 29 kDa protein band and a reduction in the rate of phosphate turnover.