A Protein Factor that Inhibits ATP-Dependent Glutamate and γ -Aminobutyric Acid Accumulation into Synaptic Vesicles: Purification and Initial Characterization

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into and stored in synaptic vesicles. We have purified to apparent homogeneity a protein from brain cytosol that inhibits glutamate and γ-aminobutyric acid uptake into synaptic vesicles and have termed this protein "inhibitory protein factor" (IPF). IPF refers to three distinct proteins with relative molecular weights of 138,000 (IPF α), 135,000 (IPF β), and 132,000 (IPF γ), respectively. Gel filtration and sedimentation data suggest that all three proteins share an elongated structure, identical Stokes radius (60 å), and identical sedimentation coefficient (4.3 S). Using these values and a partial specific volume of 0.716 ml/g, we determined the native molecular weight for IPF α to be 103,000. Partial sequence analysis shows that IPF α is derived from α fodrin, a protein implicated in several diverse cellular activities. IPF α inhibits ATP-dependent glutamate uptake into purified synaptic vesicles with an IC₅₀ of ≈26 nM, while showing no ability to inhibit ATP-independent uptake at concentrations up to 100 nM. Moreover, IPF α inhibited neither norepinephrine uptake into chromaffin vesicles nor Na⁺-dependent glutamate uptake into synaptosomes. However, IPF α inhibited uptake of γ-aminobutyric acid into synaptic vesicles derived from spinal cord, suggesting that inhibition may not be limited to glutamatergic systems. We propose that IPF could be a novel component of a presynaptic regulatory system. Such a system might modulate neurotransmitter accumulation into synaptic vesicles and thus regulate the overall efficacy of neurotransmission.