Purification and characterization of a novel, high molecular weight growth factor produced by human glioma cell lines

A novel, high molecular weight growth factor produced by human malignant glioma cell lines has been described (1, 2). This factor, termed glioma-derived growth factor 2 (GDGF-2), is sensitive to proteases, freeze-thawing and alkaline conditions (pH = 11). GDGF-2 is resistant to 2-mercaptoethanol, heating (100$\sp\circ$C, 5 min) and acidic conditions (pH = 2.0). It is not immunochemically related to other known growth factors, specifically, PDGF, bFGF, aFGF, VEGF, TGF$\alpha$, TGF$\beta$ or TNF. Chromatographic procedures revealed a high molecular weight activity ($>$200 kD, by gel filtration) with a pI of 6.5 (iso-electric focusing), which binds weakly to DEAE Sepharose and elutes in multiple peaks from reversed phase C4 column. From a panel of monoclonal antibodies (MAb) produced against partially purified GDGF-2 to aid purification and characterization, one MAb (20F3) significantly inhibited mitogenic activity of GDGF-2 ($>$50% inhibition, p $<$.05). Immunoaffinity chromatography with Sepharose conjugated with MAb 20F3 further aided purification of GDGF-2, which, again, eluted with multiple peaks with reversed phase. Autoradiography of 20F3 immunoprecipitates separated by SDS-PAGE revealed a protein band with a molecular weight of 170 kD. Upon SDS-PAGE analysis of purified GDGF-2 (affinity chromatography followed by reversed phase chromatography), a single protein was visualized which confirmed the molecular weight of 170 kD for GDGF-2. Mitogenic activity was recovered by electroelution of the 170 kD protein from polyacrylamide gels, which further confirmed growth factor activity associated with this high molecular weight protein. The 170 kD molecular weight of GDGF-2 was unaffected by 2 mercaptoethanol or F-glycopeptidase, indicating that the unusually large size of GDGF-2 was not due to subunits held by disulfide bonds or to extensive N-linked glycosylation.