Molecular properties of a cholinergic differentiation factor from muscle-conditioned medium

Conditioned medium by a variety of rat non-neuronal cells contains a protein involved in the differentiation of cholinergic neurons in cultures prepared from newborn rat superior cervical ganglion, from nodose ganglion, and from embryonic spinal cord. We have determined some hydrodynamic properties of this factor using as a bioassay the increase in choline acetyltransferase activity in sympathetic neurons grown for 12-15 days in the presence of the factor. The Stokes' radius, measured by molecular sieving chromatography on an Ultrogel AcA 44 column, was similar to that of ovalbumin (27.6 A). By analysis on 5-20% linear sucrose gradients made in H2O and D2O, we determined the partial specific volume (0.68 ml X g-1 and the sedimentation coefficient (2.1S). These data allowed the calculation of the molecular weight (21,000) and the frictional ratio f/fo (1.56). The elution pattern of the factor from a SynChropak CM 300 HPLC cation exchange column suggested that it was a basic protein. The activity of this factor was unaffected by heat treatment at 100 degrees C for 10 min.