In Vivo and In Vitro Studies on γ-Aminobutyric Acid Metabolism with the Radish Plant (Raphanus sativus, L.)

Abstract

Labeled glutamate was rapidly converted to y-aminobutyrate in intact, excised radish (Raphanus sativus L., var. Champion) leaves. Labeled y-aminobutyrate was metabolized via succinate and the Krebs cycle and was not carboxylated to form glutamate. Administration of carbon-14 and tritium-labeled succinate indicated that less than 10% of the y-aminobutyrate formation occurs by amination of succinic semialdehyde. Therefore, most -y-aminobutyrate formation must be via glutamate decarboxylation.Radish leaf extracts were more active in catalyzing transamination between y-aminobutyrate and pyruvate than that between -y-aminobutyrate and a-ketoglutarate. The striking accumulation of GAB2 in plant tissue exposed to anaerobic conditions led us to an investigation of the mechanisms responsible for this accumulation (29). Since there are two different views of the aerobic metabolism of GAB in plants, this subject was reinvestigated. One view ( Fig. 1) is that GAB is formed by decarboxylation of glutamate and is metabolized to succinate via succinic semialdehyde (2, 4, 5, 16).The alternate view is that GAB is an intermediate in the formation of glutamate from succinate (11,12,26,27), and this view has been used to explain changes in amino acid content of plants which occurred under various conditions (6,12,18,20).A more complete review of GAB metabolism and the development of these opposing viewpoints can be found elsewhere (28). This paper reports results supporting the first view (Fig. 1). Radioactive compounds which were fed to intact, excised leaves included uniformly labeled L-glutamic acid-`4C (180 tc//imole), succinic acid-2,3-'4C (10.4 jtc/ umole), succinic acid-2,3-3H (106 ,uc//mole), and y-aminobutyric acid-1-"4C (2.32 Itc//imole) (from New England Nuclear Corporation3).One microcurie in 0.1 ml of distilled water was fed to each leaf except where noted. Radioactive solutions were administered to the leaves via the petiole (30). After the radioactive solution was absorbed (15 + 5 min), distilled water was supplied. Incubations were carried out in the dark at room temperature.Analytical Methods. After incubation, leaves were thoroughly extracted with 75% (v/v) ethanol at room temperature. The alcohol extracts were dried in vacuo at 40 C or less, and the dried sample was dissolved in chloroform and water. An aliquot of the aqueous phase was put through tandem columns (0.9 X 7 cm) of Dowex 50-X8 (200-400 mesh) in the hydrogen form and of Dowex 1-X8 (200-400 mesh) in the formate form at 2 to 4 C. After washing the columns with water, we separated the columns and amino acids were eluted from the Dowex 50 with 2 N NH4OH and 40 ml of water. A partial elution of organic acids from Dowex 1 was achieved with 60 ml of 1.5 N formic acid. The eluate contained 98% or more of the succinate and malate and most of the citrate. Fumarate, pyruvate, and a-ketoglutarate were not eluted. Eluates were dried in a stream of air at room temperature.Amino acids were separated by two-directional chromatography (31) on Schleicher and...

Ammonium Assimilation and the Role of [gamma]-Aminobutyric Acid in pH Homeostasis in Carrot Cell Suspensions

“…GDC, GAB-T, and SSADH have pH optima of 5.7, 8.9, and 9.0, respectively (Streeter and Thompson, 1972b), and therefore, the GABA shunt could act as a biochemical pH-stat in a way similar to that described for malic enzyme and PEP carboxylase (Davies, 1986). In addition, SSADH is believed to be inactivated under anaerobic conditions contributing to the accumulation of GABA (Satya Narayan and Nair, 1990).…”

“…The pH-activity curves of GDC and GAB-T (Streeter and Thompson, 1972b) show that GAB-T activity is approximately 45% of its maximum value at pH,, of 7.5, whereas GDC activity is only about 4% of its maximum value (Streeter and Thompson, 1972b). Decreasing the pH to 7.3 reduces GAB-T activity only slightly but increases the GDC activity 3-fold.…”

“…Decreasing the pH to 7.3 reduces GAB-T activity only slightly but increases the GDC activity 3-fold. GDC activity has also been shown to be 20 to 30 times greater than that of GAB-T (Streeter and Thompson, 1972b;Wallace et al, 1984;Satya Narayan and Nair, 1986), and therefore, it is likely that the control of GABA accumulation would be mediated primarily via GDC.…”

“…It has also been suggested that the high accumulation of GABA in many tissues could be the result of a change in the relative activities of GDC and GAB-T following a decrease in pH,, (Streeter and Thompson, 1972b;Wallace et al, 1984). Because GDC activity increases as pH decreases and because glutamate decarboxylation is a protonconsuming reaction, it follows that GABA production could act as a sink for excess protons in the cytoplasm and thus contribute to pH regulation (Murphy et ai., 1983;Reid et al, 1985;Menegus et al, 1989;Roberts et al, 1992;Snedden et al, 1992).…”

“…It is mainly produced by the action of GDC on glutamate and it can be further metabolized to succinic semialdehyde and succinate via GAB-T and SSADH (Streeter and Thompson, 1972b;Satya Narayan and Nair, 1986). It has been suggested that the accumulation of GABA can be used as a marker for dead or dying hypoxic cells (Roberts et al, 1992).…”