The action of ␥-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H 2 O 2 and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the crossreaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H 2 O 2 exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell.

Two distinct cDNA clones encoding NAD(H)-dependent glutamate dehydrogenase (NAD[H]-GDH) in Arabidopsis thaliana were identified and sequenced. The genes corresponding to these cDNA clones were designated GDH1 and GDH2. Analysis of the deduced amino acid sequences suggest that both gene products contain putative mitochondrial transit polypeptides and NAD(H)- and α--ketoglutarate-binding domains. Subcellular fractionation confirmed the mitochondrial location of the NAD(H)-GDH isoenzymes. In addition, a putative EF-hand loop, shown to be associated with Ca2+ binding, was identified in the GDH2 gene product but not in the GDH1 gene product. GDH1 encodes a 43.0-kD polypeptide, designated α-, and GDH2 encodes a 42.5-kD polypeptide, designated [beta]. The two subunits combine in different ratios to form seven NAD(H)-GDH isoenzymes. The slowest-migrating isoenzyme in a native gel, GDH1, is a homohexamer composed of α- subunits, and the fastest-migrating isoenzyme, GDH7, is a homohexamer composed of [beta] subunits. GDH isoenzymes 2 through 6 are heterohexamers composed of different ratios of α- and [beta] subunits. NAD(H)-GDH isoenzyme patterns varied among different plant organs and in leaves of plants irrigated with different nitrogen sources or subjected to darkness for 4 d. Conversely, there were little or no measurable changes in isoenzyme patterns in roots of plants treated with different nitrogen sources. In most instances, changes in isoenzyme patterns were correlated with relative differences in the level of α- and [beta] subunits. Likewise, the relative difference in the level of α- or [beta] subunits was correlated with changes in the level of GDH1 or GDH2 transcript detected in each sample, suggesting that NAD(H)-GDH activity is controlled at least in part at the transcriptional level.

Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58-and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35-and 13-fold, respectively, by Ca 2؉ /calmodulin but not by Ca 2؉ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mM NH 4 Cl, 5 mM NH 4 NO 3 , 5 mM glutamic acid, or 5 mM glutamine as the sole nitrogen source compared with samples from plants treated with 10 mM KNO 3 . The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.

No abstract

The activity of the pyruvate dehydrogenase complex (PDC), as controlled by reversible phosphorylation, was studied in situ with mitochondria oxidizing dfifferent substrates. PDCs from both plant and animal tissues were inactivated when pyruvate became limiting. The PDC did not inactivate in the presence of saturating levels of pyruvate. Calcium stimulated reactivation of PDC in chicken heart but not pea (Pisum sativum L.) leaf mitochondria. the reactivation (dephosphorylation) of PDC using pea leaf mitochondria. This is in contrast to a previous report ( 11) with the partially purified complex where Ca2" inhibited reactivation.MATERIALS AND METHODS Materials. Pea (Pisum sativum L., cv Little Marvel) seedlings were grown in a growth chamber (10 h photoperiod, 600 uE m-2 s ') at 18°C for 2 weeks before harvesting. Intact pea leaf mitochondria were isolated and purified (devoid ofchlorophyll) using two consecutive discontinuous Percoll gradients (7). The pea leaf mitochondrial PDC is fully active as isolated. Intact potato (Solanum tuberosum) tuber mitochondria were isolated and purified as described (12). Mitochondria were prepared from the hearts ofthree 2-week-old chickens using a collagenase-facilitated isolation procedure (20).Assays. Oxygen uptake was measured at 25°C using a Hansatech electrode in a medium consisting of 20 mm Tes-KOH (pH 7.5, unless indicated otherwise), 0.3 M mannitol, 1 mm Na2EDTA, 3 mM MgCl2, 5 mM potassium phosphate (adjusted to pH 7.5), 0.1% (w/v) defatted BSA, and 10 mM KCI. The reaction mixture contained 0.1 to 0.2 mg/mL of mitochondrial protein in a final volume of 2.5 mL. Cofactor and substrate additions were performed at the times indicated and 0.2 mL aliquots were withdrawn at various times for the immediate assay of PDC activity. PDC was assayed spectrophotometrically (340 nm) at 22°C in 65 mm Tes-NaOH (pH 7.6) with 0.1I% (v/v) Triton X-100, 0.5 mM MgCl2, 2 mm (3-NAD, 0.2 mM TPP, 0.12 mM LiCoA, 1 mM L-cysteine, and 1 mm Na pyruvate. Assays were initiated with enzyme. Protein was determined by the method of Bradford (1) using BSA as the standard.The effect of calcium on PDH kinase and PDH-P phosphatase was analyzed in vitro by following the inactivation-reactivation cycle of PDC (3). Mitochondria were disrupted by addition of 0.01% (v/v) Triton X-100 in 10 mm Tes-NaOH (pH 7.5), 0.2% (w/v) defatted BSA, 1 mI MgCl2, 0.2 mM Na2EDTA, and plus/ minus 0.5 mM EGTA. Inactivation was initiated by the addition of 0.25 mM ATP.The formation of pyruvate from malate was determined by quenching 0.4 mL of the oxygen electrode reaction mix with 0.1 mL of 1.6 M HC104 in a 1.5 mL microfuge tube. The pH was neutralized with 0.2 mL of saturated KHCO3. After centrifugation, the supernatant was assayed spectrophotometrically (340 nm) for pyruvate in the presence of 50 mM Hepes-NaOH (pH 7.2), 1 mM Na2EDTA, 0.2 mM ,B-NADH, and 1 unit of lactate dehydrogenase.Phosphorylation of the PDH 43 kD subunit was analyzed on 8 to 12% gradient SDS-polyacrylamide gels. Autoradiographs were made on Kodak X-Omat...

Glyoxysomes isolated from castor-bean (Ricinus communis L.) endosperm were treated with water, 0.2 M KCl, 1 M KCl, or 0.1 M Na2CO3. Glyoxysomal sacs, i.e. membranes which retained some visible matrix, resulted from the treatments with water and KCl. Glyoxysomal ghosts, i.e. intact membranes free of matrix, were only obtained following treatment with carbonate. The ghosts were free of activities of matrix enzymes, particularly palmitoyl-CoA oxidation, isocitrate dehydrogenase (EC 1.1.1.42) and isocitrate lyase (EC 4.1.3.1), and contained only negligible amounts of malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37), β-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.98) and catalase (EC 1.11.1.6). Distribution and appearance of membrane-associated particles in the protoplasmic and ectoplasmic faces of freeze-fracture replicas of the glyoxysomal membrane were the same in intact tissue, isolated glyoxysomes, and ghosts. Membranes purified by treatment with 0.2 M KCl or 0.1 M carbonate catalyzed the reduction of cytochrome-c when NADH or NADPH was provided as the electron donor. β-Oxidation, localized in the matrix, could be linked to reduction of cytochrome-c or ferricyanide when purified membranes were combined with the matrix supernatant. Cytochrome-c could also be reduced by coupling enzyme activities in the matrix, NADP-isocitrate dehydrogenase or malate dehydrogenase, with those of the membrane. These results indicate that electrons from β-oxidation, malate oxidation or isocitrate oxidation can be transferred directly to the redox components of the glyoxysomal membrane. We, therefore, conclude that any NADH and NADPH formed by enzymes in the matrix can be recycled continuously within the organelle.

In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon Km and K1 relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [19871 Plant Physiol 83: 306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or Lcamitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.The mPDC' occupies a crucial branch point in intermediary metabolism, and activity is regulated by several mechanisms including compartmentation, product and metabolite inhibition, and reversible phosphorylation (reviewed in refs. 13,17). Initial studies of the regulatory properties of plant mPDC have been conducted in vitro using preparations that had been purified 20-to 300-fold (18-21). Recently, we (2-4, 16) have developed a method for assaying mPDC activity in situ using purified intact pea seedling mitochondria. Using this method, several observations on the regulation by reversible phosphorylation have been clarified and extended (2-4). This report continues our investigation of the role of product inhibition in regulation of PDC activity within intact mitochondria.Much like mammalian and microbial PDCs, plant mPDCs

ABSTRACIGlyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720 and 140 nanomole electrons/per minute per milligram glyoxysomal protein, respectively. These redox activities were greater than could be attributed to contmination of the glyoxysomal fractions in which 1.4% of the protein was mitochondrial and 5% endoplasmic reticulum. The NADH:ferricyanide reductase activity in the glyoxysomes was greater than the palmitoyl-coenzyme A (CoA) oxidation activity which generated NADH at a rate Malate was also oxidized by glyoxysomes, if acetyl-CoA, ferricyanide, or cytochrome c was present. Glyoxysomal NADH:ferricyanide reductase activity has the capacity to support the combined rates of NADH generation by 8-oxidation and the glyoxylate cycle.