Glutamate-glyoxylate Aminotransferase Research Articles

Exogenous methyl jasmonate promotes the biosynthesis of endogenous melatonin in mustard sprouts

The present study investigated the effects regulating melatonin (MT) biosynthesis under methyl jasmonate (MeJA) treatment in mustard sprouts. The results revealed that MeJA significantly increased the MT content in the sprouts to 11.43 times that of the control. However, MeJA treatment had an inhibitory effect on growth. Tryptophan decarboxylase and tryptamine 5-hydroxylase gene expression were significantly induced by MeJA. Moreover, 156 differential abundance proteins (DAPs) were detected in 4-day-old sprouts using quantitative proteomic methods. These DAPs were divided into 13 functional groups, and the vast majority of DAPs involved in defense/stress, energy, signal transduction, and secondary metabolism increased. MeJA treatment significantly enriched 15 pathways, including glutathione metabolism, biosynthesis of secondary metabolites, and tryptophan metabolism. In particular, the abundance of three DAPs (myrosinase 1, cytosolic sulfotransferase 16, and glutamate-glyoxylate aminotransferase 2) in the tryptophan metabolism pathway, a substrate for MT biosynthesis, increased significantly. In summary, MeJA induces endogenous MT biosynthesis in mustard sprouts by promoting the genes expression of MT synthetase and increasing the abundance of tryptophan-related proteins.

Metagenomics and Metabolomics Study on Microbial Diversity and Enzymatic Activities Changes on Cow Dung Based Low-Temperature Biogas Production by Entererococcus spp

To fill the gap between the rise in demand for energy and decline in the traditional energy sources such as coal, natural gas and nuclear energy, other alternative sources such as biogas are necessary. Studies have shown that the existing conditions within the fermentation realm control the microbial characteristics in biogas production. However, there is inadequate insight between the duration of fermentation and the microbial diversity, and with specific emphasis to cow manure as the substrate under low temperature fermentation. This study aimed at providing additional insight on the effect of varying fermentation duration (0 to 60 days) on the composition of the dominant microbial flora on cow dung based low-temperature (15 °C) biogas fermentation using metagenomics and metabolomics analyses approach. The study results showed that the main dominant community in the process of methanogenesis are the Spirochaetae, Synergistetes and Chloroflexi, and are new flora in the methane phase. In the peak stage of low-temperature biogas fermentation, the dominant bacteria groups were Methanosarcina and after adding 10% concentration of L1 bacteria. The prediction of metabolic pathway was mainly carbohydrate metabolism and amino acid metabolism with succinyl-CoA synthase a subunit, lactaldehyde reductase and the glutamate-glyoxylate aminotransferase being the main unique enzymes. The study therefore supports the potential of involving the reported dominant microbial communities and related enzymatic activities for improved biogas production under low temperature conditions.

Coupling physiological analysis with proteomic profile to understand the photosynthetic responses of young Euterpe oleracea palms to drought.

This study examined whether drought sensitivity in açaí (Euterpe oleracea Mart.) is associated with reductions in photosynthesis and increasing oxidative stress in response to down-regulation of proteins related to photosynthetic reactions, photorespiration, and antioxidant system. Well-watered (Control) and drought-stressed plants were compared when leaf water potential in stressed plants reached around - 1.5 and - 3.0MPa, representing moderate and severe drought. Drought caused 84 and 96% decreases in net photosynthetic rate (Pn) and stomatal conductance. Stress-mediated changes in maximum quantum efficiency of photosystem II (PSII) photochemistry were unobserved, but drought decreased photochemical quenching, actual quantum yield of PSII electron transport, and apparent electron transport rate (ETR). Moderate and severe drought induced, respectively, decreases and increases in non-photochemical quenching (NPQ) and 74 and 273% increases in ETR/Pn. Moderate drought down-regulated PSII protein D2, chlorophyll a-b binding protein 8, photosystem I reaction center subunit N, sedoheptulose-1,7-bisphosphatase, and transketolase; while severe drought down-regulated LHC II proteins, ferredoxin-NADP reductase, ATP synthase subunits ε and ß, and carbonic anhydrase isoform X2. The glutamate-glyoxylate aminotransferase 2 and glycine dehydrogenase were down-regulated upon moderate drought, while catalase 2 and glycine cleavage system H protein 3 were up-regulated. Severe drought up-regulated glycolate oxidase, glycine cleavage system H protein 3, and aminomethyl transferase, but most of photorespiration-related proteins were only found in control plants. Down-regulation of chaperones and antioxidant enzymes and increased lipid peroxidation in stressed plants were observed upon both stress severities. Therefore, the decreases in Pn and failure in preventing oxidative damages through adjustments in NPQ and photorespiration- and antioxidant-related proteins accounted for drought sensitivity in açaí.

Nitrogen-mediated alleviation of photosynthetic inhibition under moderate water deficit stress in rice (Oryza sativa L.)

High nitrogen (N) application improves the acclimation of plant photosynthesis to water deficit stress, however, the metabolic details for this are not yet fully understood. Using hydroponics, we found that moderate water deficit simulated with 10% PEG 6000 inhibited rice photosynthetic rate by 17.2% in low N condition (LN, 0.71 mM) versus 7.9% in moderate N condition (MN, 2.86 mM). Plants grown at MN had an increased photorespiratory serine metabolism and glycerate recycling under water stress, which was in accordance with the enhancement of glutathione concentration and ascorbate-glutathione cycle, indicating that the serine metabolism plays a significant role in improving antioxidant capacity. Additionally, the up-regulation of GS2 facilitated the re-assimilation of NH3 released in photorespiration. Aspartate aminotransferase (AspAT)- and glutamate:glyoxylate aminotransferase (GGAT)-mediated glutamate transamination and phosphoenolpyruvate carboxylase (PEPC)-mediated anaplerotic reaction provided a carbon skeleton, 2-oxoglutarate, for NH3 assimilation, which reduced the depletion of sugars. Sugars therefore can be stored and used for the regeneration of RuBP through the pentose phosphate pathway to maintain CO2 assimilation. In contrast, water deficit stress-induced protein degradation, down-regulation of N assimilation, and depletion of carbohydrates in LN-supplied plants, as well as the failure of the ascorbate-glutathione cycle due to reduced glutathione biosynthesis, perturbed the function of chloroplasts. We conclude that high N supply preserves the biochemistry of photosynthesis through coordinated regulation of the C and N metabolism to facilitate the acclimation of rice photosynthesis to water deficit stress.

Drought stress impact on leaf proteome variations of faba bean (Vicia faba L.) in the Qinghai\u2013Tibet Plateau of China

Water scarcity is a major abiotic stress factor that strongly influences growth, development and yield of grain legumes in arid and semi-arid area of the world. Drought stress frequently occurs during the seedling stage and finally affects yield of faba bean (Vicia faba L.). However, the responses of plant leaf to drought have not been documented very well at the proteomic level. “Ga da dou” of the drought-tolerant faba bean cultivar was exposed to drought to examine the proteome changes of leaves. In this study, 2-week-old seedlings were subjected to water deficit by 7 days drought stress, whereas control plants were regularly irrigated. After withdrawing water, plants exposed to drought for 7 days and control plants at the same developmental stage were included in quantitative proteomic analysis using two-dimensional electrophoresis gels of proteins in combination with mass spectrometry. Over 300 proteins were detected by 2-DE, 50 differentially expressed proteins were detected by t test and 30 proteins were successfully identified by MALDI-TOF/TOF, in which 25 proteins were clearly downregulated and five proteins were upregulated. The quantified proteins were grouped into five functional groups, mainly regulatory proteins (46.7%), energy metabolism (23.3%), cell cytoskeleton (6.7%), other functions (20%) and unknown function (3.3%). Chitinase was upregulated under drought, suggesting that it was an important part of the plant defense system, playing an important role in stress resistance. 50S ribosomal protein was upregulated under drought, suggesting its role in protecting plants against stress by re-establishing normal protein conformations. The abundance of proteins involved in protein synthesis such as chitinase, Bet protein and glutamate–glyoxylate aminotransferase was upregulated under drought stress. These proteins could play important roles in drought tolerance and contribute to the relatively stronger drought tolerance of “Ga da dou”.

Comparison of leaf proteomes of potato (Solanum tuberosum L.) genotypes with ER- and HR-mediated resistance to PVY infection

The Ny-1 and Ry-fsto genes of potato (Solanum tuberosum L.) confer hypersensitive response (HR) and extreme resistance (ER), respectively, to Potato virus Y (PVY). ER-type resistance was also observed in potato plants with both alleles (Ny-1 and Ry-fsto). Using two-dimensional electrophoresis (2-DE), quantitative differences between PVY-infected and non-infected control plants were found for 35, 32 and 15 protein spots identified in leaves of potato cultivar Rywal (possessing Ny-1), and potato tetraploid clones PW 363 (with Ry-fsto) and PB 08–137 (with Ny-1 + Ry-fsto), respectively. We recognized 29, 12 and 21 PVY-induced protein spots involved in qualitative changes in Rywal, PW 363 and PB 08–137 plants, respectively, which were processed and analysed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. A database search indicated that these 62 proteins belong to various functional categories. Their expression was genotype-specific. In the case of cultivar Rywal with HR-mediated resistance, proteins involved in photosynthesis and primary metabolism were the most abundant. For PW 363 and PB 08–137, both with ER–mediated resistance, stress-responsive proteins were the most numerous. Only two proteins – glutamate–glyoxylate aminotransferase 2 (GGAT2) and monodehydroascorbate reductase 5 (MDHAR5) – were identified in all three genotypes. These two proteins are components of the reactive oxygen species (ROS) defence mechanism against pathogens in plants. The present study showed that the differences in PVY-induced proteins in the leaves of Ny-1, Ry-fsto and Ny-1 + R-fsto genotypes do not correspond to the type of gene conferring the resistance or to the observed phenotype.

Identification and expression analyses of the alanine aminotransferase (AlaAT) gene family in poplar seedlings

Alanine aminotransferase (AlaAT, E.C.2.6.1.2) catalyzes the reversible conversion of pyruvate and glutamate to alanine and α-oxoglutarate. The AlaAT gene family has been well studied in some herbaceous plants, but has not been well characterized in woody plants. In this study, we identified four alanine aminotransferase homologues in Populus trichocarpa, which could be classified into two subgroups, A and B. AlaAT3 and AlaAT4 in subgroup A encode AlaAT, while AlaAT1 and AlaAT2 in subgroup B encode glutamate:glyoxylate aminotransferase (GGAT), which catalyzes the reaction of glutamate and glyoxylate to α-oxoglutarate and glycine. Four AlaAT genes were cloned from P. simonii × P. nigra. PnAlaAT1 and PnAlaAT2 were expressed predominantly in leaves and induced by exogenous nitrogen and exhibited a diurnal fluctuation in leaves, but was inhibited in roots. PnAlaAT3 and PnAlaAT4 were mainly expressed in roots, stems and leaves, and was induced by exogenous nitrogen. The expression of PnAlaAT3 gene could be regulated by glutamine or its related metabolites in roots. Our results suggest that PnAlaAT3 gene may play an important role in nitrogen metabolism and is regulated by glutamine or its related metabolites in the roots of P. simonii × P. nigra.

Distinct photorespiratory reactions are preferentially catalyzed by glutamate:glyoxylate and serine:glyoxylate aminotransferases in rice

The metabolic function of glutamate:glyoxylate aminotransferase (GGAT) and serine:glyoxylate aminotransferase (SGAT) for photorespiration is still not exactly understood so far though it is mostly held that both enzymes may work in parallel in the reaction of glyoxylate to glycine during photorespiration of plants. Here, for the first time, we define the genes encoding GGAT and SGAT and report their biochemical and enzymatic properties in rice plants, in contrast to those from other plant species. Noticeably, GGAT exhibited approximately 18 fold higher catalytic efficiency (Kcat/Km) with glyoxylate and glutamate than SGAT with glyoxylate and serine, and additionally, rice leaves usually contain 3–4times higher abundance of glutamate relative to serine, implicating that GGAT may preferentially utilize glyoxylate to form glycine over SGAT. When SGAT or GGAT activity was regulated by gene transformation or nitrogen deficiency, respectively, it was observed that the glycine content was positively related to GGAT activities, while both serine and glycine contents were negatively related to SGAT activities. The results suggest that GGAT preferentially catalyzes the conversion of glyoxylate into glycine while SGAT is mainly responsible for the transamination reaction of serine to hydroxypyruvate in the photorespiratory pathway of rice.

Photorespiration Process and Nitrogen Metabolism in Lettuce Plants (Lactuca sativa L.): Induced Changes in Response to Iodine Biofortification

Iodine is vital to human health, and iodine biofortification programs help improve human intake through plant consumption. There is no research on whether iodine biofortification influences basic plant physiological processes. Because nitrogen (N) uptake, utilization, and accumulation are determining factors in crop yield, the aim of this work was to establish the effect of the application of different doses (20, 40, and 80 μM) and forms of iodine (iodate [IO3 −] vs. Iodide [I−]) on N metabolism and photorespiration. For this study we analyzed shoot biomass and the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), glutamate dehydrogenase (GDH), glycolate oxidase (GO), glutamate:glyoxylate aminotransferase (GGAT), serine:glyoxylate aminotransferase (SGAT), hydroxypyruvate reductase (HR) and catalase (CAT), nitrate (NO3 −), ammonium (NH4 +), organic and total N, amino acids, proteins, serine (ser), malate, and α-ketoglutaric acid in edible lettuce leaves. Application of I− at doses of at least 40 μM reduced the foliar concentration of NO3 − with no decrease in biomass production, which may improve the nutritional quality of lettuce plants. In contrast, the application of 80 μM of I− is phytotoxic for lettuce plants, reducing the biomass, foliar concentration of organic N and NO3 −, and NR and GDH activities. HR activity is significantly inhibited with all doses of I−; the least inhibition was at 80 μM. This may involve a decrease in the incorporation of carbonated skeletons from photorespiration into the Calvin cycle, which may be partially associated with the biomass decrease. Finally, the application of IO3 − increases biomass production, stimulates NO3 − reduction and NH4 + incorporation (GS/GOGAT), and optimizes the photorespiratory process. Hence, this appears to be the most appropriate form of iodine from an agronomic standpoint.

Glyoxylate rather than ascorbate is an efficient precursor for oxalate biosynthesis in rice

Oxalate is widely distributed in the plant kingdom. While excess oxalate in food crops is detrimental to animal and human health, it may play various functional roles in plants, particularly for coping with environmental stresses. Understanding its biosynthetic mechanism in plants, therefore, becomes increasingly important both theoretically and practically. However, it is still a matter of debate as to what precursor and pathway are ultimately used for oxalate biosynthesis in plants. In this study, both physiological and molecular approaches were applied to address these questions. First, it was observed that when glycolate or glyoxylate was fed into detached leaves, both organic acids were equally effective in stimulating oxalate accumulation. In addition, the stimulation could be completely inhibited by cysteine, a glyoxylate scavenger that forms cysteine-glyoxylate adducts. To verify the role of glyoxylate further, various transgenic plants were generated, in which several genes involved in glyoxylate metabolism [i.e. SGAT (serine-glyoxylate aminotransferase), GGAT (glutamate-glyoxylate aminotransferase), HPR (hydroxypyruvate reductase), ICL (isocitrate lyase)], were transcriptionally regulated through RNAi or over-expression. Analyses on these transgenic plants consistently revealed that glyoxylate acted as an efficient precursor for oxalate biosynthesis in rice. Unexpectedly, it was found that oxalate accumulation was not correlated with photorespiration, even though this pathway is known to be a major source of glyoxylate. Further, when GLDH (L-galactono-1,4-lactone dehydrogenase), a key enzyme gene for ascorbate biosynthesis, was down-regulated, the oxalate abundance remained constant, despite ascorbate having been largely reduced as expected in these transgenic plants. Taken together, our results strongly suggest that glyoxylate rather than ascorbate is an efficient precursor for oxalate biosynthesis, and that oxalate accumulation and regulation do not necessarily depend on photorespiration, possibly due to the occurrence of the anaplerotic reaction that may compensate for glyoxylate formation in rice.

Assay of alanine:glyoxylate aminotransferase in human liver by its serine: glyoxylate aminotransferase activity

We have examined assay methods and conditions for human liver alanine:glyoxylate aminotransferase (AGT). This enzyme shows not only the AGT activity but also serine:pyruvate and serine: glyoxylate aminotransferase (SPT and SGT) activities. In the assay of AGT activity using crude enzyme preparations, there is the complication that glutamate:glyoxylate aminotransferase (GGT) also contributes to AGT activity, but at present no other enzyme is known to catalyze transamination between L-serine and glyoxylate or pyruvate. Therefore, an assay for AGT using its SGT activity was investigated in which hydroxypyruvate formed from L-serine in the enzymic reaction with glyoxylate was determined by lactate dehydrogenase (LDH) in the presence of tris(hydroxym ethyl)aminomethane (Tris) at pH 8.4. A possible obstacle to this assay is that pyruvate formed from L-serine by serine dehydratase (SDH) interferes with SGT assay as an additional substrate of LDH and AGT. However, the SDH activity in human liver is very low, and by performing the SGT reaction in the presence and absence of glyoxylate the SGT activity was represented as the glyoxylate-dependent hydroxypyruvate formation from L-serine. There was a combined good correlation between the AGT, SGT and SPT activities, and the activity ratio, AGT : SGT : SPT was about 1.0 : 0.17 : 0.13.

Glutamate:Glyoxylate Aminotransferase Modulates Amino Acid Content during Photorespiration

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.

Characterization of alanine aminotransferase (AlaAT) multigene family and hypoxic response in young seedlings of the model legume Medicago truncatula

Four alanine aminotransferases (AlaATs) are expressed in Medicago truncatula. In adult plants, two genes encoding mitochondrial isoforms m-AlaAT and alanine-glyoxylate aminotransferase (AGT), catalysing, respectively, reversible reactions of alanine/oxoglutarate<==>glutamate/pyruvate and alanine/glyoxylate<==>glycine/pyruvate, were expressed in roots, stems, and leaves. A gene encoding a cytosolic (c-AlaAT) isoform, catalysing the same reaction as m-AlaAT, was expressed specifically in leaves, while a gene encoding an isoform involved in branched chain amino acid metabolism was expressed in stems and roots. In young seedlings, only m-AlaAT and AGT were expressed in embryo axes. In hypoxic embryo axes, the amounts of transcript and putative protein of m-AlaAT (EC 2.6.1.2) increased while those of AGT (EC 2.6.1.44) decreased and in vivo enzyme activities changed as revealed by [(15)N]alanine and [(15)N]glutamate labelling. Under hypoxia, m-AlaAT catalysed only alanine synthesis while glutamate synthesis using alanine as amino donor was inhibited. As a result, alanine accumulated as the major amino acid in hypoxic seedlings instead of asparagine, in agreement with the involvement of the fermentative AlaAT pathway in hypoxia tolerance. Regulation of m-AlaAT at both the transcriptional and post-translational levels allowed for an increase in gene expression and orientation of the activity of the product of its transcription towards alanine synthesis under hypoxia. Labelling experiments showed that glycine synthesis occurred at the expense of either alanine or glutamate as amino donor, indicating that a glutamate-glyoxylate aminotransferase was operating together with AGT in Medicago truncatula seedlings. Both enzymes seemed to be inhibited by hypoxia, resulting in a very low amount of glycine in hypoxic seedlings.

Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis.

In the photorespiratory process, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutarate and glycine. Although GGAT has been assumed to play important roles for the transamination in photorespiratory carbon cycles, the gene encoding GGAT has not been identified. Here, we report that an alanine:2-oxoglutarate aminotransferase (AOAT)-like protein functions as GGAT in peroxisomes. Arabidopsis has four genes encoding AOAT-like proteins and two of them (namely AOAT1 and AOAT2) contain peroxisomal targeting signal 1 (PTS1). The expression analysis of mRNA encoding AOATs and EST information suggested that AOAT1 was the major protein in green leaves. When AOAT1 fused to green fluorescent protein (GFP) was expressed in BY-2 cells, it was found to be localized to peroxisomes depending on PTS1. By screening of Arabidopsis T-DNA insertion lines, an AOAT1 knockout line (aoat1-1) was isolated. The activity of GGAT and alanine:glyoxylate aminotransferase (AGAT) in the above-ground tissues of aoat1-1 was reduced drastically and, AOAT and glutamate:pyruvate aminotransferase (GPAT) activity also decreased. Peroxisomal GGAT was detected in the wild type but not in aoat1-1. The growth rate was repressed in aoat1-1 grown under high irradiation or without sugar, though differences were slight in aoat1-1 grown under low irradiation, high-CO2 (0.3%) or high-sugar (3% sucrose) conditions. These phenotypes resembled those of photorespiration-deficient mutants. Glutamate levels increased and serine levels decreased in aoat1-1 grown in normal air conditions. Based on these results, it was concluded that AOAT1 is targeted to peroxisomes, functions as a photorespiratory GGAT, plays a markedly important role for plant growth and the metabolism of amino acids.

Primary hyperoxaluria type 1

Primary hyperoxaluria type 1

Aminooxy acetic acid: a selective inhibitor of alanine:glyoxylate aminotransferase and its use in the diagnosis of primary hyperoxaluria type I

The diagnosis of primary hyperoxaluria type I is usually based on the determination of the activity of the hepatic peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). The activity observed, however, is due to the action of at least one more enzyme, i.e. glutamate:glyoxylate aminotransferase (GGT). Correction for the AGT activity displayed by GGT is usually made by use of a correction factor which correlates the activity of GGT with the amount of ‘AGT’ activity exhibited by GGT. This method, however, has a number of drawbacks: it corrects for only one other enzyme and it requires a second, rather insentitive and laborious enzyme assay to be performed which cannot be adapted to a centrifugal analyser. We therefore developed a simple and direct method for measurement of ‘true’ AGT activity which uses 100 μmol/l aminooxy acetic acid. Under these conditions AGT is completely inhibited and the contribution of GGT (and possibly other transaminases) to the l-alanine mediated transamination of glyoxylate can be measured directly. The ‘true’ AGT activities measured by this method correlated well with those measured in samples depleted of AGT by immunoprecipitation. Finally, this method proved to be fully compatible with the automated assay of AGT in a centrifugal analyser.

Isolation and characterization of peroxisomes from diatoms

Peroxisomes were isolated by gradient centrifugation from two different diatoms: Nitzschia laevis (subgroup of Pennales) and Thalassiosira fluviatilis (subgroup of Centrales). In neither of these organelles could catalase or any H2O2-forming oxidase be demonstrated. The glycolate-oxidizing enzyme present in the peroxisomes is a dehydrogenase capable of oxidizing l-lactate as well. The peroxisomes also contain the glyoxysomal markers isocitrate lyase and malate synthase. However, enzymes of the fatty-acid β-oxidation pathway are located exclusively in the mitochondria. The mitochondria additionally possess glutamate-glyoxylate aminotransferase and a glycolate dehydrogenase which differs from the peroxisomal glycolate dehydrogenase since it preferably utilizes d-lactate as an alternative substrate. Hydroxypyruvate reductase and glyoxylate carboligase were not found in the cells of either diatom. By culturing Nitzschia laevis it could be demonstrated that decreasing the CO2 concentration in the aeration mixture from 2% to 0.03% and increasing the irradiance from 40 to 250 μmol quanta · m−2 · s−1 resulted in an increase of all peroxisomal enzyme activities. In addition, enzyme activities of the β-oxidation pathway were increased. However, mitochondrial glycolate dehydrogenase and aminotransferase did not alter their activities under these conditions. Summarizing all results, it is postulated that there are two different pathways for the metabolism of glycolate in the diatoms.

Compartmentation Studies on Spinach Leaf Peroxisomes

In concurrence with earlier results, the following enzymes showed latency in intact spinach (Spinacia oleracea L.) leaf peroxisomes: malate dehydrogenase (89%), hydroxypyruvate reductase (85%), serine glyoxylate aminotransferase (75%), glutamate glyoxylate aminotransferase (41%), and catalase (70%). In contrast, glycolate oxidase was not latent. Aging of peroxisomes for several hours resulted in a reduction in latency accompanied by a partial solubilization of the above mentioned enzymes. The extent of enzyme solubilization was different, being highest with glutamate glyoxylate aminotransferase and lowest with malate dehydrogenase. Osmotic shock resulted in only a partial reduction of enzyme latency. Electron microscopy revealed that the osmotically shocked peroxisomes remained compact, with smaller particle size and pleomorphic morphology but without a continuous boundary membrane. Neither in intact nor in osmotically shocked peroxisomes was a lag phase observed in the formation of glycerate upon the addition of glycolate, serine, malate, and NAD. Apparently, the intermediates, glyoxylate, hydroxypyruvate, and NADH, were confined within the peroxisomal matrix in such a way that they did not readily leak out into the surrounding medium. We conclude that the observed compartmentation of peroxisomal metabolism is not due to the peroxisomal boundary membrane as a permeability barrier, but is a function of the structural arrangement of enzymes in the peroxisomal matrix allowing metabolite channeling.

Subcellular Distribution of Enzymes of Glycolate Metabolism in the Alga Cyanidium caldarium

The intracellular distribution of enzymes capable of catalyzing the reactions from phosphoglycolate to glycerate in the bluegreen colored eucaryotic alga Cyanidium caldarium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes glycolate oxidase and glutamate-glyoxylate aminotransferase along with catalase were present in the peroxisomal fraction (density: 1.23 grams per cubic centimeter). Serine hydroxymethyltransferase was found in the mitochondrial fraction (density: 1.18 grams per cubic centimeter). In contrast to the observations in green leaves of higher plants, the enzymes for the conversion of serine to glycerate (serine-glyoxylate aminotransferase and hydroxypyruvate reductase) were found only in the soluble fraction of the gradient. The partial characterization of enzymes from Cyanidium participating in glycolate metabolism revealed only slight differences from the corresponding enzymes from higher plants. The phylogenetic implications of the observed similarities between the enigmatic alga Cyanidium and higher plants are discussed.

Compartmentation of Peroxisomal Enzymes in Algae of the Group of Prasinophyceae

Using the Prasinophycean algae Platymonas, Heteromastix, Pedinomonas, and Pyramimonas, subcellular distribution of the enzymes of glycolate metabolism and beta-oxidation pathway have been studied. Glycolate dehydrogenase, hydroxypyruvate reductase, and glutamate-glyoxylate aminotransferase are located in the mitochondria. In addition, the mitochondria of all four species contain acyl-coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and thiolase. In Platymonas, Heteromastix, and Pedinomonas, organelles with the characteristic structure of peroxisomes have been detected which also contain the enzymes acyl-CoA oxidase, enoyl-CoA hydratase and thiolase. However, catalase could not be demonstrated in either the peroxisome-like organelles or in the whole cells.