A New Synaptosomal Biosynthetic Pathway of Glutamate and GABA from Ornithine and Its Negative Feedback Inhibition by GABA

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Abstract
In sonicates of mouse brain synaptosomes, we demonstrated that γ-aminobutyric acid (GABA) can be formed from l-ornithine (Orn) through l-glutamic acid (Glu), but not through putrescine (Put). Incubation of these sonicates with [3H]ORN yielded not only [3H]Glu and [3H]l-proline(Pro) but also produced [3H]GABA from the [3H]Glu. Formation of each of these three major amino acids from [3H]Orn was strongly inhibited by the addition of GABA (1-5 mM). The likely enzymatic site of this negative feedback inhibition by GABA appeared to be ornithine δ-amhotransferase (OAT). A radiometric procedure was employed to study the effects of the three amino acids cited above and of others found in the free form in brain on the activity of a 30-fold-purified OAT from rat brain. Enzyme activity was measured in the presence of low concentrations of Orn, such as might occur in vivo. OAT was inhibited by GABA to a considerably greater extent than by Glu, l-glutamine, or Put; no inhibition was found with Pro, glycine, aspartarte, taurine, or β-alanine. The inhibition by GABA was competitive with Orn. These results clearly show that one of the molecular mechanisms underlying the negative feedback inhibition of synaptosomal GABA biosynthesis from Om is a competitive inhibition by GABA of the brain OAT activity that is responsible for the formation of l-glutamic-γ-semialdehyde ⇌l-Δ1-pyrroline-5-carboxylicacid from Orn. Thus, the results suggest that GABA may play an important role in restricting the metabolic flow from Orn to Glu and thence to GABA. It was confirmed that l-canaline (δ-aminooxy-l-α-aminobutyric acid) is a potent and specific inhibitor of brain OAT, whereas much weaker inhibition was observed with two other carbonyl-trapping agents, aminooxyacetic acid and hydrazine.