Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions.
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.
Chin
G J
GJ
Howard Hughes Medical Institute, Center for Neurobiology and Behavior, Columbia University, New York, New York 10032.
Shapiro
E
E
Vogel
S S
SS
Schwartz
J H
JH
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
D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D011494:Protein Kinases