Isolation of small agranular synaptic vesicles of rat brain by gel filtration chromatography.
I attempted to isolate synaptic vesicles by gel filtration. The rat brain synaptic vesicles in a synaptosomal lysate were collected by ammonium sulfate salting-out and fractionated on a Sephacryl S-500 with a mean exclusion size of 200 nm. Peak I at the void volume contained large vesicular membranes and coated vesicles besides synaptic vesicles; Peak II consisted almost entirely of small agranular synaptic vesicles of 40-50 nm diameter; and Peak III comprised soluble proteins. Western blotting revealed that components of 72 kDa in peaks I and II reacted with an anti-H(+)-ATPase A-subunit antibody [Moriyama et al. (1995) FEBS Lett. 367, 233-236]. When examined for Mg(2+)-ATPase activity, peak I showed specific activity of 4.52 ( micromol ATP hydrolyzed/mg protein/30 min), while that of peak II was as low as 0.22. As estimated from the inhibition by bafilomycin A(1) [Bowman et al. (1988) PROC: Natl. Acad. Sci. USA 85, 7972-7976], the percentage of H(+)-ATPase as to total Mg(2+)-ATPase, 18-22%, was unchanged, indicating no accumulation of the H(+)-ATPase in peak II even on the chromatography. In brief, the small agranular synaptic vesicles in peak II showed little or no Mg(2+)-ATPase activity, although they reacted with the H(+)-ATPase antibody. The reason for this is obscure. Mg(2+)-ATPase might not be a constituent of small agranular synaptic vesicles of rat brain.
Tsudzuki
Toshihiro
T
Department of Biochemistry I, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-0001.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D008854:Microscopy, Electron; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013347:Subcellular Fractions / Q000378:metabolism; D013572:Synaptic Vesicles / Q000737:chemistry* / Q000648:ultrastructure
D004791:Enzyme Inhibitors; D018942:Macrolides; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D000254:Sodium-Potassium-Exchanging ATPase