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. Author manuscript; available in PMC: 2013 Apr 1.

Published in final edited form as: J Neurochem. 2012 Mar 13;121(2):184–196. doi: 10.1111/j.1471-4159.2012.07684.x

Fig. 8 — The α-KGA-derived glutamate uptake system is the most abundant in the subcellular fraction, the richest in synaptic vesicles and devoid of mitochondrial contamination. (a) α-KGA-derived glutamate uptake. Various subcellular fractions were analyzed for ATP-dependent α-KGA-derived glutamate uptake in the presence of 60 μM [¹⁴C]α-KGA, using 75 μg protein. SV-A, SV-A′, SV-B, SV-B′, SV-C, SV-C′, and D: synaptic vesicle (SV) subfractions obtained upon sucrose density-gradient centrifugation of the crude synaptic fraction, as described in Materials and Methods. Values are the mean ± SEM of 4 or 5 separate preparations. (b) Relative contents of VGLUT1 and synaptophysin1 in various synaptic vesicle fractions and plasma membranes. Various synaptic vesicle fractions and the plasma membrane fraction, 4 μg each, were subjected to SDS/western blotting, and probed with antibodies against VGLUT1 (1:1000 dilution) and synaptophysin1 (1:5000 dilution). Glu, glutamate; PM, plasma membrane plus mitochondrial fraction. (c) Relative contents of cytochrome oxidase subunit IV in various synaptic vesicle fractions and the mitochondria fraction. Various synaptic vesicle fractions and the mitochondria fraction, 10μg each, were subjected to SDS/western blotting, and probed with antibodies against cytochrome oxidase subunit IV (1:5000 dilution). Mit, mitochondria; A, SV-A; A′, SV-A′; B, SV-B; B′, SV-B′; C, SV-C; C′, SV-C′; D, pellet.

Fig. 8 — The α-KGA-derived glutamate uptake system is the most abundant in the subcellular fraction, the richest in synaptic vesicles and devoid of mitochondrial contamination. (a) α-KGA-derived glutamate uptake. Various subcellular fractions were analyzed for ATP-dependent α-KGA-derived glutamate uptake in the presence of 60 μM [¹⁴C]α-KGA, using 75 μg protein. SV-A, SV-A′, SV-B, SV-B′, SV-C, SV-C′, and D: synaptic vesicle (SV) subfractions obtained upon sucrose density-gradient centrifugation of the crude synaptic fraction, as described in Materials and Methods. Values are the mean ± SEM of 4 or 5 separate preparations. (b) Relative contents of VGLUT1 and synaptophysin1 in various synaptic vesicle fractions and plasma membranes. Various synaptic vesicle fractions and the plasma membrane fraction, 4 μg each, were subjected to SDS/western blotting, and probed with antibodies against VGLUT1 (1:1000 dilution) and synaptophysin1 (1:5000 dilution). Glu, glutamate; PM, plasma membrane plus mitochondrial fraction. (c) Relative contents of cytochrome oxidase subunit IV in various synaptic vesicle fractions and the mitochondria fraction. Various synaptic vesicle fractions and the mitochondria fraction, 10μg each, were subjected to SDS/western blotting, and probed with antibodies against cytochrome oxidase subunit IV (1:5000 dilution). Mit, mitochondria; A, SV-A; A′, SV-A′; B, SV-B; B′, SV-B′; C, SV-C; C′, SV-C′; D, pellet.