Purification of stalk‐cell‐inducing morphogens from Dictyostelium discoideum

We have shown previously that developing amoebae of Dictyostelium discoideum release one or more low‐ M r factors, which can induce isolated cells to differentiate into stalk cells in the presence of cyclic AMP [Town, C. D., Gross, J. D. and Kay, R. R. (1976) Nature (Lond.) 262 , 717–719; Town, C. D. and Stanford, E. (1979) Proc. Natl Acad. Sci. USA, 76 , 308–312]. These differentiation‐inducing factors (DIF) have now been purified by a procedure involving binding to and elution from XAD‐2 resin, extraction into hexane and two steps of reverse‐phase high‐pressure liquid chromatography (HPLC). Our results show the following.1 HPLC resolves a major stalk‐cell‐inducing activity (DIF‐1) and at least four minor and more polar activities (DIFs 2–5). 2 DIF‐1 has been purified at least 3000‐fold over the starting dialysed medium with a recovery of about 2%. This low recovery of DIF‐1 can be explained in part by the loss of non‐specific stimulatory (‘helper’) factors during the purification. A few micrograms purified DIF‐1 were obtained from 10 12 cells. This material could induce stalk cell differentiation in the standard assay at less than 0.2 nM. 3 The biological activity of DIFs 1, 2 and 3 was sensitive to borohydride reduction, suggesting the presence of an essential carbonyl group. DIF‐5 was partially sensitive and DIF‐4 resistant. Other properties of DIF‐1 suggest that it is a non‐polar molecule of M r < 500, which becomes charged in alkaline solution, and that it is neither a peptide nor has essential sugar moieties. 4 The purification of DIF should make possible its eventual identification by sensitive physical techniques, such as mass spectroscopy, and will allow further investigation of its biological effects.