| Abstract: | The subcellular distribution of pyridoxal phosphate (PLP) was studied in mouse brain, as well as the effect of pyridoxal phosphate-γ-glutamyl hydrazone (PLPGH—a convulsant drug which decreases both PLP levels and glutamate decarboxylase activity GAD] in whole brain) upon both the PLP concentration and the GAD activity in subcellular fractions. An electron microscopic evaluation of the subcellular particles of control and PLPGH-treated animals was also carried out. The main findings were the following: (1) PLP was localized mainly in the supernatant and crude mitochondrial fractions; two-thirds of the amount present in the latter were located in the subfraction containing pure mitochondria, and the remainder was in the synaptosomal fraction. After osmotic disruption of synaptosomes, PLP was found in both the intrasynaptosomal mitochondria and the synaptoplasm. (2) Treatment of mice with PLPGH decreased levels of PLP in several brain fractions, this effect being much more notable in the soluble fractions than in the particulate fractions. After osmotic disruption of the synaptosomes, a specific decrease of PLP in the synaptoplasm was observed. (3) Treatment with PLPGH produced also an inhibition of GAD activity in most of the fractions studied, when this enzyme was assayed in the absence of PLP. In general, the inhibition was greater in those fractions in which levels of PLP were also affected. In synaptosomes, this correlation between the decreased levels of PLP and decreased activity of GAD occurred only in the synaptoplasm. (4) The activation of GAD by PLP added to incubation mixtures was much greater in those fractions from PLPGH-treated animals which displayed extensive inhibition of GAD, in comparison to the corresponding fractions from control animals. (5) No ultrastructural changes were detected in the subcellular fractions from treated animals. Our results show that the decreases of both the levels of PLP and the activity of GAD (as previously found in whole brain) actually occur in the synaptosomes, a finding that supports the hypothesis that the role of PLP in the mechanisms controlling excitability can be explained, at least in part, by its regulatory action on GAD activity, which in turn determines the rate of GABA synthesis at the nerve endings. |
