Purification of melanoma growth stimulatory activity

The Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low‐density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum‐free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse‐phase high‐pressure liquid chromatography (RP‐HPLC), and preparative electrophoresis, resulting in a >400,000‐fold purification. MGSA bioactivity resides in acid‐ and heat‐stable polypeptides of high and low molecular weight (24–28 kd and < 14–16 kd). However, the majority of the activity is reproducibly associated with the ˜16‐kd moiety eluting from RP‐HPLC at ∼35% acetonitrile. Reduction with dithiothreitol or B‐mercaptoethanol results in a loss of biological activity but does not convert the 24–28‐kd moieties to the