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Metabolism and Enzymology

The Production and Efflux of 4-Aminobutyrate in Isolated Mesophyll Cells ¹

Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada, L2S 3A1, Department of Horticultural Sciences, University of Guelph, Guelph, Ontario, Canada, N1G 2W1

The pathway of 4-aminobutyric acid (GABA) production and efflux was investigated in suspensions of mesophyll cells isolated from asparagus (Asparagus sprengeri Regel) cladophylls. Analysis of free amino acids demonstrated that, on a molar basis, GABA represented 11.4, 19, and 6.5% of the xylem sap, intact cladophyll tissue, and isolated mesophyll cells, respectively. L-Glu, a GABA precursor, was abundant in intact cladophylls and isolated cells but not in xylem sap. When cells were incubated with L-[U-¹⁴C]Glu, intracellular GABA contained less than 10% of the radioactivity found in intracellular Glu. However, GABA in the medium contained 78% of the radioactivity found in extracellular L-Glu metabolites. Incubation with L-[1-¹⁴C]Glu resulted in the appearance of unlabeled GABA, demonstrating its production through decarboxylation at carbon 1. GABA released to the medium from cells incubated with L-[U-¹⁴C]Glu had a specific activity of 18 nanocuries per nanomole, whereas GABA remaining in the cell had a specific activity of 2.25 x 10^–1 nanocuries per nanomole. In the presence of exogenous L-Glu, amino acid analysis and cell volume measurements indicated intracellular Ala and GABA concentrations of 4.2 and 1.4 millimolar, respectively. In the medium, however, the corresponding concentrations were 2 and 57 micromolar. The data indicate that L-Glu entering the cell is decarboxylated to GABA, and that specific and passive efflux is from this pool of recently synthesized GABA and not from a previously synthesized unlabeled pool of GABA.

¹ Supported by grants A4453 and UO343 from the Natural Sciences and Engineering Research Council of Canada to A.W.B. and B.J.S.

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