Osmotic fragility of chromaffin granules prepared under isoosmotic or hyperosmotic conditions and localization of acetylcholinesterase.
In chromaffin cells of the adrenal medulla, catecholamines are stored in secretory granules. Different methods have been described to purify chromaffin granules. In the present study, storage granules were prepared using isoosmotic self-generating Percoll gradients or hyperosmotic sucrose gradients, and a comparison of their physical properties in response to osmotic changes was made. Catecholamines, dopamine beta-hydroxylase activity and protein were detected both in the external medium and in the granule fraction according to the medium osmolality. Suspension turbidity was used as a measure of organelle integrity. Acetylcholinesterase activity was found to be associated with both isoosmotically and hyperosomotically prepared granules. The total acetylcholinesterase activity was determined after adding Triton X-100 to the assay medium. When adrenal medullary tissue was homogenized in buffers containing echothiopate, an inhibitor of acetylcholinesterase, only 15-20% of enzyme activity was inhibited, excluding the possibility that main granule acetylcholinesterase could be due to contamination by plasma membrane fragments, endoplasmic reticulum and Golgi membranes. When granules were suspended in hypoosmotic buffers, a soluble acetylcholinesterase form was released into the external medium, while an insoluble acetylcholinesterase form was still found associated with the membrane fraction. Soluble acetylcholinesterase was found to be released differently than soluble dopamine beta-hydroxylase, indicating that acetylcholinesterase may be associated with a more osmotically resistant granule population.
D000110:Acetylcholinesterase / Q000032:analysis*; D000818:Animals; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002837:Chromaffin Granules / Q000201:enzymology / Q000502:physiology*; D002838:Chromaffin System / Q000502:physiology*; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism; D009994:Osmolar Concentration
D002395:Catecholamines; D002800:Cholinesterase Inhibitors; D004299:Dopamine beta-Hydroxylase; D000110:Acetylcholinesterase