l-Glutamate-Dependent Medium Alkalinization by Asparagus Mesophyll Cells

Abstract

Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of Lglutamate or its analog L-methionine-D,L-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas L-aspartate did not. Fusicoccin stimulated Ho efflux from the cells by 111% and the uptake of L-[U-'4Cjglutamate by 55%.Manometric experiments demonstrated that, unlike L-methionine-D,Lsulfoximine, L-glutamate stimulated CO2 evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and "CO2 evolution upon the addition of labeled L-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas "CO2 evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of L-IU-'4Cjglutamate were higher in the light while rates of '4CO2 evolution were higher in the dark. The major labeled product of glutamate decarboxylation, y-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO2 and -y-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H'/L-glutamate uptake process.A plasma membrane-located, ATP-driven, H+-efflux process establishes a H+ electrochemical gradient across the membrane of plant cells (22 an alkalinization of the suspension medium (7,12,13,16,18,19,25). However, alkalinization may also arise from metabolism of the transported amino acid (18). Protonation of a basic metabolite will result in alkalinization as will a decarboxylation process:RCOO-+ H+--RH + CO2 On the basis of medium alkalinization and uptake studies, an H+ cotransport system specific for L-Glu3 and L-MSO has been proposed for photosynthetically competent Asparagus mesophyll cells (12). However, the apparent H+ uptake rate was 5 to 8 times faster than the uptake of L-["4C]Glu indicating a large molar excess of H+ associated with the L-Glu uptake process. This investigation also demonstrated that addition of L-Glu results in the rapid release of CO2 (12). These findings indicate that the consumption of H+ may result from a metabolic process. Medium alkalinization would occur if L-Glu was decarboxylated by an extracellular enzyme prior to the entry of the decarboxylated product into the cell. This model is analogous to the hydrolysis of sucrose by an extracellular invertase prior to hexose cotransport into sugarcane cells (8). Medium alkalinization would also occur if glutamate cotransport into the cell resulted in the export and protonation of a basic product of L-Glu metabolism. Alternatively, the high H+ to L-Glu ratio could arise from the loss of labeled CO2 or other metabolites from the cell an...

Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells

“…(15), far higher than theoretical or measured stoichiometries found in other systems (20,21,28). The elevated stoichiometry resulted from an underestimation of labeled L-Glu uptake due to its decarboxylation and the rapid efflux of the labeled product GABA5 (1,15).…”

“…vessel (15 The other product of the GAD-catalyzed reaction, [14C] GABA, was identified by TLC and autoradiography. TLC plates were developed as described above, sprayed with betadecay enhancer (Dupont), and exposed to x-ray film (Kodak) at -70°C for 2 to 4 weeks.…”

“…In isolated Asparagus mesophyll cells, a H+/L-Glu symport process causes rapid alkalinization of the medium for 30 to 40 min (14,15). Calculations indicate that, without active compensating mechanisms, the cytoplasmic pH would decline by 1.9 pH units/h.…”

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

“…Attempts to determine the stoichiometry of the H+/L-G1u cotransport system by simultaneously measuring the initial rate of L-Glu-dependent alkalinization and the initial rate of L-[U-'4C]Glu uptake yielded unexpectedly high stoichiometries ranging from 3.0 to 7.7:1. Further experiments determined that a high proportion of the label entering the cells as L-[U-14C]Glu was released to the medium as labeled GABA and 14CO2 (1,10). The efflux of GABA and CO2 led to an underestimation of the rate of L-Glu entry and, consequently, to an overestimation of the H+/L-Glu stoichiometry.…”

“…A H+/L-Glu cotransport system has been characterized at the plasma membrane ofAsparagus sprengeri mesophyll cells (1,9,10). Attempts to determine the stoichiometry of the H+/L-G1u cotransport system by simultaneously measuring the initial rate of L-Glu-dependent alkalinization and the initial rate of L-[U-'4C]Glu uptake yielded unexpectedly high stoichiometries ranging from 3.0 to 7.7:1.…”