Isolation of Cholinergic Synaptic Vesicles from the Myenteric Plexus of Guinea‐Pig Small Intestine

Abstract

The acetylcholine-rich myenteric plexus-longitudinal muscle preparation of the guinea pig small intestine was subjected to subcellular fractionation, using modifications of the classical methods and methods originally devised for bulk isolation of cholinergic synaptic vesicles from the electromotor nerve terminals of Torpedo marmorata by density gradient centrifugation in a zonal rotor. The latter method gave a vesicle fraction with the highest acetylcholine content so far recorded for a mammalian particulate fraction, 30.9 .+-. standard mean of error 1.8 (5) nmol of acetylcholine .cntdot. mg of protein-1. EM examination showed that it apparently consisted of a homogeneous preparation of vesicles of mean spherical diameter 61 .+-. SD 4 (108) nm, with little or no contamination with other lipoprotein membrane structures, mixed however with considerable amounts of actomyosin fibrils, presumably derived from the longitudinal muscle. Slab-gel electrophoresis in sodium dodecyl sulfate showed that, in addition to prominent peaks attributable to actin and myosin, there apparently was a relatively simple pattern of (presumably) vesicle protein among which all the proteins thought to be characteristic of Torpedo synaptic vesicles were present.