A diffusible factor responsible for the determination of cholinergic functions in cultured sympathetic neurons. Partial purification and characterization.
A macromolecule has been partially purified which influences the choice of the neurotransmitter synthesized by sympathetic neurons. Previous studies showed that culture medium conditioned by incubation on certain types of non-neuronal cells increased [3H]acetylcholine synthesis and accumulation from [3H]choline in primary cultures of neurons dissociated from neonatal rat superior cervical ganglion and grown in the virtual absence of non-neuronal cells. A concomitant decrease of [3H]catecholamines production was observed (Patterson, P. H., and Chun, L. L. Y. (1977) Dev. Biol. 56, 263-280). The active cholinergic factor in conditioned medium from C6 glioma or primary rat heart cultures has been purified about 1500-fold by a sequence of ammonium sulfate precipitation, DEAE-, CM-cellulose, and Sephadex G-100 chromatography. The partially purified factor is active at 1 microgram of protein/ml of culture medium and is eluted from Sephadex columns as a single peak of apparent Mr = 40,000-45,000. This material is insensitive to 0.2 M 2-mercaptoethanol, but is inactivated by 1 mM Na periodate. Its activity is partially decreased by treatment with a protease from Streptomyces griseus but is unaffected by neuraminidase. Material purified through the ammonium sulfate, DEAE- and CM-cellulose steps contains large amounts of trypsin- and chymotrypsin-inhibiting activities.
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D005728:Ganglia, Sympathetic / Q000378:metabolism*; D005910:Glioma; D007700:Kinetics; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000096:biosynthesis*; D010277:Parasympathomimetics / Q000302:isolation & purification*; D051381:Rats; D014443:Tyrosine / Q000378:metabolism
D018377:Neurotransmitter Agents; D010277:Parasympathomimetics; D014443:Tyrosine; D002794:Choline