Isolation of Small Agranular Synaptic Vesicles of Rat Brain by Gel Filtration Chromatography

Abstract

I attempted to isolate synaptic vesicles by gel filtration. The rat brain synaptic vesi-cles in a synaptosomal lysate were collected by ammonium sulfate salting-out and fractionated on a Sephacryl S-500 with a mean exclusion size of 200nm. 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 40-50nm 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 Mg²⁺-ATPase activity, peak I showed specific activity of 4.52 (μmol ATP hydrolyzed/mg protein/30 min), while that of peak II was as low as 0.22. As estimated from the inhibition by bafilomycin A₁ [Bowman et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7972-7976], the percentage of H⁺-ATPase as to total Mg²⁺-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 Mg²⁺-ATPase activity, although they reacted with the H⁺-ATPase antibody. The reason for this is obscure. Mg²⁺-ATPase might not be a constituent of small agranular synaptic vesicles of rat brain.