Induction of acetylcholine receptor synthesis and aggregation: partial purification of low-molecular-weight activity

We have studied the effect of saline and acid extracts of chick brain on the total number of acetylcholine (ACh) receptors and the number of receptor clusters in cultured chick muscle cells. Myotubes in 7-day cultures responded more rapidly to brain extract than did myotubes in 4-day cultures, so the older cells were used in subsequent bioassays. A large percentage of the receptor inducing activity was soluble in 2% trifluoroacetic acid (TFA), and this material appeared by Sephadex G-25 chromatography to be about 1000 daltons in size. Activity was retained on octadecasilyl silica and was further purified by reverse-phase high-pressure liquid chromatography using a TFA-acetonitrile gradient system. Material that eluted between 35 and 40% acetonitrile, termed C4018, was 500- to 1000-fold more potent than saline extract. The receptor accumulation induced by C4018 was associated with an increased rate of receptor incorporation, presumably receptor synthesis, rather than to a decrease in receptor degradation. An increase in incorporation was detected as early as 3 hr after C4018 was added to 7-day cultures and the effect was maximal after 10 hr. C4018 also promoted the aggregation of receptors that were already incorporated in the surface membrane at the time to addition. It is not yet known if aggregation of "old" receptors and increased receptor synthesis are related or if the two phenomena are mediated by the same molecule.