Synaptic vesicles from mammalian brain: large-scale purification and physical and immunochemical characterization.
Purification of synaptic vesicles directly from homogenates of mammalian brain is compared with a classical method based on osmotic lysis of brain synaptosomes. The direct method affords increased yield and purity of synaptic vesicles prepared under isoosmotic conditions. Antigen SV2 and the antigens (primarily synaptophysin) recognized by rabbit antiserum R10, raised to purified rat brain synaptic vesicles, are localized specifically on approximately 40-nm-diameter microsomal vesicles from rat brain. Rat brain synaptic vesicles have equilibrium densities of approximately 1.11 g/ml on Nycodenz density gradients, 1.12 g/ml on glycerol/Nycodenz, and 1.07 g/ml on Ficoll gradients. Both SV2 and the R10 antigens are enriched approximately 50-fold in purified rat brain synaptic vesicles. Synaptic vesicles purified from rat or cow brain show active uptake of [3H]norepinephrine that is reserpine sensitive and dependent on ATP and Mg2+. Synaptic vesicles exhibiting [3H]norepinephrine uptake comigrate with approximately 40-nm-diameter synaptic vesicles carrying SV2 or R10 antigens during permeation chromatography. After the Sephacryl S-1000 chromatography step, [3H]-norepinephrine uptake activity is purified approximately 90-fold. Highly purified brain synaptic vesicles should facilitate studies at the molecular level of the roles of these organelles in neurotransmission at mammalian synapses.
Floor
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Department of Anatomy, University of Wisconsin Medical School, Madison 53706.
Schaeffer
S F
SF
Feist
B E
BE
Leeman
S E
SE
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D008854:Microscopy, Electron; D009638:Norepinephrine / Q000378:metabolism; D009997:Osmotic Pressure; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure*
D009638:Norepinephrine