Proton/L-glutamate symport and the regulation of intracellular pH in isolated mesophyll cells

Abstract

Addition of L-[U-14C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengen Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of L-[U-14C] glutamate, and (c) efflux of [14C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H+/L-glutamate cotransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H+/L-Glu symport may involve both H+ consumption during 4-aminobutyrate production and ATP-driven H+ efflux.