title: Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions.
abstract: We prepared and characterized subcellular membrane fractions from the CNS of Aplysia californica that are enriched in isolated nerve terminals (synaptosomes). Ganglia were homogenized in 1.1 M sucrose and fractionated on a 2-step sucrose gradient, yielding 50 micrograms protein/animal in the synaptosomal fraction (P3), which was enriched 3-fold in plasma membrane as compared with the initial homogenate. Quantitative morphometry of electron micrographs revealed that P3 contained 25% intact synaptosomes, a 5-fold enrichment over the homogenate. Although fractionation on a 5-step sucrose gradient reduced the yield of protein in the synaptosomal fraction to 40 micrograms/animal, this fraction (the 0.35 M/0.75 M interface) was more enriched in plasma membrane than P3 and was less contaminated by lysosomes and free mitochondria. By electron microscopy, the 0.35 M/0.75 M interface contained up to 50% synaptosomes. Synaptosomal fractions contained cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinase activities and were enriched in a Mr 40,000 pertussis toxin substrate, Gi/o. In the accompanying paper, we show that these synaptosomes retain the ability to release transmitters.
authors.affiliation: Howard Hughes Medical Institute, Center for Neurobiology and Behavior, Columbia University, New York, New York 10032.
authors.forename: G J
authors.initials: GJ
authors.lastname: Chin
authors.forename: E
authors.initials: E
authors.lastname: Shapiro
authors.forename: S S
authors.initials: SS
authors.lastname: Vogel
authors.forename: J H
authors.initials: JH
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D001048:Aplysia / Q000201:enzymology / Q000648:ultrastructure*; D002490:Central Nervous System / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D005724:Ganglia / Q000648:ultrastructure; D008566:Membranes / Q000648:ultrastructure; D037342:Pertussis Toxin; D011494:Protein Kinases / Q000032:analysis; D013347:Subcellular Fractions / Q000648:ultrastructure; D013574:Synaptosomes / Q000201:enzymology / Q000648:ultrastructure*; D010566:Virulence Factors, Bordetella
chemical_list: D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D011494:Protein Kinases