Purification of stalk-cell-inducing morphogens from Dictyostelium discoideum

We have shown previously that developing amoebae of Dictyostelium discoideum release one or more low-Mr 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. HPLC resolves a major stalk-cell-inducing activity (DIF-1) and at least four minor and more polar activities (DIFs 2-5). 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. 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 Mr less than 500, which becomes charged in alkaline solution, and that it is neither a peptide nor has essential sugar moieties. 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.