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J. Biochem, 2003, Vol. 133, No. 4 437-443
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

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

Toshihiro Tsudzuki+

Department of Biochemistry I, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-0001

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 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.

+ To whom correspondence should be addressed. Tel: +81-52-853-8141, Fax: +81-52-841-3480


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