Molecular and Ce&dar Endocrinology, 57 (1988) 69-76 Elsevier Scientific Publishers Ireland, Ltd. 69 MCE 01845 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA High yield pu~fication of mekmoma growth s~~~ato~ activity H. Greg Thomas and Ann Richmond V.A. Medical Center (Atlania), Department of Medicine, Division of Endocrinology, Emory University School of Medicine, Atlanta, GA 30322, U.S.A (Received 23 September 1987; accepted 13 January 1988) Key wordx Heparin-Sepharose; Melanoma; Growth factor purification Tumor cells produce a variety of hormones and growth factors that are associated with modulation of the growth pattern of malignant cells. Hs294T human malignant melanoma cells produce a monolayer mitogen, melanoma growth stimulator activity (MGSA), that stimulates the growth of Hs294T cultures in serum-free medium. MGSA has been purified to homogeneity from conditioned medium of Hs294T human malignant melanoma cells using acetic acid extraction of the crude conditioned medium followed by three chromatographic processes, including gel-filtration, heparin-Sepharose, and reverse-phase HPLC. MGSA was eluted from the heparin-Sepharose resin with 0.1-0.3 M NaCl. The binding affinity is similar to that of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) but much less than many endothelial cell-derived growth factors which require significantly higher salt concentrations for elution. These procedures resulted in a final yield of purified MGSA that was significantly greater than yields obtained using previously reported procedures. The homogeneous 16000 and 13 000 molecular weight moieties obtained by means of these procedures exhibited similar bioactivities (stimulating a 2- to 3-fold increase in Hs294T cell growth) over a 0.06-6 ng concentration range. This bioactivity w as progressively inactivated during storage at - 8O’C. These results indicate that the combination of heparin-Sepharose chromatography and reverse phase-HPLC provides a more efficient means of purifica- tion of MGSA. Introduction The culture conditions necessary to support in vitro growth of melanocytes require the use of a number of additives, including serum, 12-O-tetra- decanoylphorbol lZacetate, cholera toxin, iso- Address for correspondence: H. Greg Thomas, V.A. Medi- cal Center (Atlanta), Department of Medicine, Division of Endocrinology, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. Supported by NC1 Grant CA34590, NIH Training Core Grant DK07298 and V.A. Merit Award. butyl-rncthy~~t~e (Eisinger and Marko, 1982; Halaban and Alfano, 1984) and extracts of cells or tissues such as melanoma cells, astrocytoma cells, WI-38 cultured fibroblasts (Eisinger et al., 1985) and bovine brain (Wilkins et al., 1985). Cultured malignant melanoma cells do not require the pres- ence of these additives (Halaban et al., 1986). This observation led us to examine the hypothesis that malignant melanoma cells produce endogenous autostimulatory growth factors (Richmond et al., 1982a). Work from this laboratory has shown that culture medium conditioned by malignant mela- noma cells or extracts of melanoma cells contain 0303-7207/88/$03.50 6 1988 Elsevier Scientific Publishers Ireland, Ltd.