Purification of a growth-suppressing factor for bovine artery endothelial cells from mouse lymphoma P388D1 cells

Abstract

A growth-suppressing factor for bovine artery endothelial cells (BAEGSF) was purified from the conditioned medium of a mouse lymphoma P388D1 cell culture in the presence of 100 μg/ml carboxymethylated curdlan. The purification steps included, in order, ammonium sulfate fractionation and eight stages of column chromatography on Macro-Prep Ceramic Hydroxyapatite, Q-Sepharose, Sephacryl S-300 HR, Matrex PBA-30, CHT II, Resource-Q, anti- bovine serum albumin (BSA) agarose, and Superdex 200HR columns. The purified BAEGSF showed two bands with silver staining on a sodium dodecyl sulfate polyacrylamide gel under reducing conditions (SDS-PAGE) and their molecular weights were estimated as approximately 55 and 63 kDa, while the molecular weight of the purified BAEGSF was estimated as about 65 kDa by gel filtration using Superdex 200HR. This result shows that BAEGSF obtained from Superdex 200HR chromatography is a partially purified preparation and suggests that one of the two bands on SDS-PAGE corresponds to BAEGSF. BAEGSF was shown not to have a lethal effect on endothelial cells, but had an inhibitory action on the proliferation of these cells. Furthermore, the growth-suppressing activity of BAEGSF for bovine artery endothelial cells (BAE) was not inhibited by anti-transforming growth factor-β (TGF-β), anti-tumor necrosis factor-α (TNF-α), and anti-interleukin-1 (IL-1) antibodies. These results suggest that BAEGSF is different from TGF-β, TNF-α, and IL-1 which have been reported to inhibit BAE growth.