Figure 2.

Vesicular uptake of l-aspartate is independent of sialin. A) Measurement of ATP-dependent vesicular accumulation of ³H-l-aspartate in whole-brain synaptic vesicles from sialin-knockout (KO) and wild-type (WT) mice. ATP-dependent uptake of l-aspartate was 9.5 ± 1.8 pmol/min/mg protein in the WT and 13.8 ± 5.2 pmol/min/mg protein in the KO (means±se, n=4 experiments, P>0.05, Student's t test; SPSS). B) Western blots of vesicles derived from WT (WT PC12) and PC12 cells expressing recombinant sialin (A3) with an antibody directed against sialin (left panel) and RFP (right panel) demonstrate marked overexpression of sialin in the stable cell line. The mCherry tag (RFP) on the recombinant protein causes the primary band in the recombinant cells to migrate more slowly than the band in the WT cells. C) Measurement of vesicular accumulation (means±sd; nmol/mg/5 min, n=2 experiments) of ³H-l-aspartate (open bars) and ³H-l-glutamate (solid bars) in crude synaptic-like microvesicles derived from wild-type PC12 cells (WT PC12) and those expressing recombinant sialin (A3 sialin). There is a >3-fold increase in accumulation of l-glutamate in synaptic-like microvesicles from VGLUT1 (B1 VGLUT1)-expressing cells compared to WT. Brackets indicate that the glutamate uptake was significantly higher in B1 VGLUT1 than in WT (P<0.05, Student's t test).