Serine-glyoxylate Aminotransferase Research Articles

A mutant of Nicotiana sylvestris deficient in serine glyoxylate aminotransferase activity

A photorespiration mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity was isolated in the M2 generation following EMS mutagenesis. Mutants showing chlorosis in air and normal growth in 1% CO2 were fed [(14)C]-2-glycolate to examine the distribution of (14)C among photorespiratory intermediates. Mutant strain NS 349 displayed a 9-fold increase in serine accumulation relative to wild-type controls. Enzyme assays revealed an absence of serine: glyoxylate aminotransferase (SGAT) activity in NS 349, whereas other peroxisomal enzymes were recovered at normal levels. Heterozygous siblings of NS 349 segregating air-sensitive M3 progeny in a 3∶1 ratio were shown to contain one half the normal level of SGAT activity, indicating that air sensitivity in NS 349 results from a single nuclear recessive mutation eliminating SGAT activity. Since toxicity of the mutation depends on photorespiratory activity, callus cultures of the mutant were initiated and maintained under conditions suppressing the formation of functional plastids. Plantlets regenerated from mutant callus were shown to retain the SGAT deficiency and conditional lethality in air. The utility of photorespiration mutants of tobacco as vehicles for genetic manipulation of ribulose bisphosphate carboxylase/oxygenase at the somatic cell level is discussed.

Effect of cyclic amp on suncus murinus liver serine-aminotransferase and serine dehydratase

1. 1. Suncus murinus was injected dibutyryl adenosine 3′,5′-cyclic monophosphate (Bt 2cAMP) and assayed serine-glyoxylate aminotransferase (EC 2.6.1.45) and serine dehydratase (EC 4.2.1.13). 2. 2. Serine dehydratase was induced 4-fold by Bt 2cAMP. The K m values of the induced enzyme for l-serine and pyridoxal 5′-phosphate was 57 mM and 3.0 μM, respectively. The enzyme had a pH optimum at pH 10.0. These kinetic properties and pH optimum were same as those of the enzyme from the control. Both the holoenzyme and the apoenzyme increased to the same extent by Bt 2cAMP. 3. 3. Serine-glyoxyate aminotransferase activity was decreased slightly by the Bt 2cAMP injection. The holoenzyme activity was increased, but the apoenzyme decreased. K m values for l-serine and glyoxylate of this enzyme were 6mM and 0.2 mM, respectively, without change by Bt 2cAMP.

Light-stimulated accumulation of the peroxisomal enzymes hydroxypyruvate reductase and serine:glyoxylate aminotransferase and their translatable mRNAs in cotyledons of cucumber seedlings

The development of peroxisomal enzymes in cotyledons of cucumber seedlings is strongly dependent on light. In light-grown seedlings, activities of two peroxisomal enzymes, hydroxypyruvate reductase (HPR) and serine: glyoxylate aminotransferase (SGAT), were barely detectable until three days postimbibition, after which time both activities increased rapidly and linearly for at least three days. In the dark, the activities of these enzymes increased slightly over the same time period, but only to about 5% to 10% of 7-day light-induced levels. When 51/2-day dark-grown seedlings were transferred into white light, activities of HPR and SGAT began to increase after approximately 8 h. HPR protein was shown by an immunoprecipitation assay to increase concurrently with enzymatic activity in both light- and dark-grown cotyledons. Immunoblotting results suggested that the amounts of SGAT-A and SGAT-B, the two subunits of SGAT, also developed along with SGAT activity. The relative levels of translatable mRNAs encoding HPR, SGAT-A, and SGAT-B were also light-dependent, and increased with a developmental pattern similar to enzyme activity and protein levels in light- and dark-grown cotyledons. In 51/2-day dark-grown cotyledons that were transferred to the light, translatable mRNAs for SGAT-A and SGAT-B began to increase within 1 h of illumination and continued of increase rapidly and linearly for the next 24 h in the light to a new steady-state level that was 45 times that of dark controls. Translatable HPR mRNA exhibited a biphasic pattern of accumulation, with a three-fold increase during the first 6 h of illumination, followed by an additional six-fold increase between 8 and 24 h. The accumulation of translationally active mRNA for both enzymes preceded the accumulation of the corresponding protein and enzyme activity by about 8 h. Our data suggest that the rise in enzyme activity depends on an increase in translatable mRNA for these enzymes and is regulated at a pretranslational level, most likely involving transcription of new mRNA.

Conversion of serine to glycerate in intact spinach leaf peroxisomes: Role of malate dehydrogenase

In photorespiration, leaf peroxisomes convert serine to glycerate via serine-glyoxylate aminotransferase and NADH-hydroxypyruvate reductase. We isolated intact spinach leaf peroxisomes in 0.25 m sucrose, and characterized their enzymatic conversion of serine to glycerate using physiological concentrations of substrates and coenzymes. In the presence of glycolate (glyoxylate), and NADH and NAD alone or together in physiological proportions, the rate of serine-to-glycerate conversion was enhanced and sustained by the addition of malate. The rate was similar at 1 and 5 m m serine, but was two to three times higher in 50 m m than 5 m m malate. In the presence of NAD and malate, there was 1:1 stoichiometric formation of glycerate and oxaloacetate. Addition of 1 or 5 m m glutamate resulted in a negligible enhancement of the conversion of hydroxypyruvate to glycerate. Intact peroxisomes produced glycerate from either serine or hydroxypyruvate at a rate two times higher than osmotically lysed peroxisomes. These results suggest that under physiological conditions, the peroxisomal malate dehydrogenase operates independent of aspartate-α-ketoglutarate aminotransferase in supplying NADH for hydroxypyruvate reduction. This supply of NADH is the rate-limitng step in the conversion of serine to glycerate. The compartmentation of hydroxypyruvate reductase and malate dehydrogenase in the peroxisomes confers a higher efficiency in the supply of NADH for hydroxypyruvate reduction under a normal, high NAD NADH ratio in the Cytosol.

Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy.

Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function. We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A-gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.

Photorespiration-Induced Reduction of Ribulose Bisphosphate Carboxylase Activation Level

Leaf photosynthesis and ribulose bisphosphate carboxylase activation level were inhibited in several mutants of the C(3) crucifer Arabidopsis thaliana which possess lesions in the photorespiratory pathway. This inhibition occurred when leaves were illuminated under a photorespiratory atmosphere (50% O(2), 350 microliters per liter CO(2), balance N(2)), but not in nonphotorespiratory conditions (2% O(2), 350 microliters per liter CO(2), balance N(2)). Inhibition of carboxylase activation level was observed in strains with deficient glycine decarboxylase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, glutamate synthase, and chloroplast dicarboxylate transport activities, but inhibition did not occur in a glycolate-P phosphatase-deficient strain. Also, the photorespiration inhibitor aminoacetonitrile produced a decline in leaf and protoplast ribulose bisphosphate carboxylase activation level, but was without effect on intact chloroplasts. Fructose bisphosphatase, a light-activated enzyme which is strongly dependent on stromal pH and Mg(2+) for regulation, was unaffected by conditions which caused inhibition of ribulose bisphosphate carboxylase. Thus, the mechanism of inhibition does not appear to involve changes in stromal Mg(2+) and pH but rather is associated with metabolite flux through the photorespiratory pathway.

Glycolate Metabolism Is Under Nitrogen Control in Chlorella

The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR).During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate.

Control of photorespiratory glycolate metabolism in an oxygen-resistant mutant of Chlorella sorokiniana.

Under a gas atmosphere of 99% O2/1% CO2, wild-type cells of Chlorella sorokiniana excreted 12% of their dry weight as glycolate during photolithotrophic growth, whereas mutant cells excreted glycolate at only 3% of the cellular dry weight. The observed difference in glycolate excretion by the two cell types appears to be due to a different capacity for the metabolism of glycolate, rather than to a different glycolate formation rate. This was concluded from experiments in which the metabolism of glycolate via the glycine-serine pathway was inhibited by the addition of isoniazid. Under such conditions, glycolate excretion rates for both cell types were identical. The mutant appeared to have significantly higher specific activities of glycine decarboxylase, serine hydroxymethyltransferase, serine-glyoxylate aminotransferase, glycerate kinase, and phosphoglycolate phosphatase than did the wild type. The specific activities of D-ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate dehydrogenase, glyoxylate-aminotransferase, and hydroxypyruvate reductase were the same for wild-type and mutant cells. The internal pool sizes of ammonia and amino acids increased in wild-type cells grown under high-oxygen concentrations but were hardly affected by high oxygen tensions in the mutant cells. Our results indicate that, under the growth conditions applied, the decarboxylation of glycine becomes the rate-limiting step of the glycine-serine pathway for the wild-type cells of C. sorokiniana.

Purification and Characterization of Hydroxypyruvate Reductase from Cucumber Cotyledons

Hydroxypyruvate reductase (HPR), a marker enzyme of peroxisomes, has been purified to homogeneity from cotyledons of light-grown cucumber seedlings (Cucumis sativus var. Improved Long Green). In addition, the peroxisomal location of both HPR and serine-glyoxylate aminotransferase has been confirmed in cucumber cotyledons. The isolation procedure involved Polymin-P precipitation, a two-step precipitation with ammonium sulfate (35 and 50% saturation), affinity chromatography on Cibacron Blueagarose, and ion-exchange chromatography on DEAE-cellulose. HPR was purified 541-fold to a final specific activity of 525 +/- 19 micromoles per minute per milligram of protein. Enzyme homogeneity was established by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was 91 to 95 kilodaltons, approximately double the apparent subunit molecular weight of 40,500 +/- 1,400. With hydroxypyruvate as substrate, the pH optimum was 7.1 and K(m) values were 62 +/- 6 and 5.8 +/- 0.7 micromolar for hydroxypyruvate and NADH, respectively. With glyoxylate as substrate, the pH optimum was 6.0, and the K(m) values for glyoxylate and NADH were 5700 +/- 600 and 2.9 +/- 0.5 micromolar, respectively. Antibodies to HPR were raised in mice (by the ascites tumor method) and in rabbits, and their monospecificity was demonstrated by a modified Western blot immunodetection technique.

Purification and properties of an asparagine aminotransferase from Pisum sativum leaves

The enzyme responsible for the transamination of l-asparagine in pea leaves has been partially purified. It appears to be the same protein as the serine-glyoxylate aminotransferase. It is able to use serine or asparagine as amino donors and pyruvate or glyoxylate as amino acceptors. The reaction is reversible but the equilibrium is toward glycine or alanine production. The favored substrates are serine and glyoxylate: serine shows competitive inhibition toward asparagine, as does pyruvate toward glyoxylate. Substrate interaction and product inhibition patterns are consistent with a pingpong mechanism. The enzyme has a pH optimum at 8.1. Gel filtration indicates a M r of 105,000. Inhibition was caused by aminoxyacetate and hydroxylamine, but the enzyme was unaffected by isonicotinic acid hydrazide. The apoenzyme was resolved and was inactive: addition of pyridoxal 5′-phosphate restored 85% of the original activity.

Metabolism of glycolate in Euglena gracilis. Part V. Occurrence and subcellular distribution of enzymes involved in the glycolate pathway and their physiological function in a bleached mutant of Euglena gracilis Z.

The streptomycin-bleached, non-photosynthetic mutant of Euglena gracilis contained NADH-glyoxylate reductase, NADPH-glyoxylate reductase, glutamate-glyoxylate aminotransferase and serine-glyoxylate aminotransferase, in addition to glycolate dehydrogenase. Most activities of NADH- and NADPH-glyoxylate reductases and 75% of glutamate-glyoxylate aminotransferase activity in the crude homogenate were recovered in mitochondria.The mutant cells grown under illumination contained three times as high glycolate dehydrogenase activity as the dark-grown cells. Illumination on the dark-grown cells caused an increase in activity to the level of the light-grown cells. Light at 420nm wavelength was most effective for inducing the enzyme. The enzyme induced by illumination was the mitochondrial one and the specific activity increased 4-fold after illumination for 3 days; the rate of the increase was almost equal to the ratio of the rate of glycolate uptake from incubation medium by the light-grown mutant to that of the d...

Asparagine and glutamine metabolism in Rhodopseudomonas acidophila.

Rhodopseudomonas acidophila strain 7050 achieved balance growth when provided with either asparagine or glutamine as nitrogen source. Under these growth conditions R. acidophila synthesized a mixed amidase which exhibited similar activity (223--422 nmol/min . mg protein) against either nitrogen source. Determination of the free intracellular amino acid pools show that deamidation of asparagine and glutamine resulted in elevated levels of both aspartate and glutamate. Cell-free extracts of R. acidophila showed significant aminotransferase activity, particularly glutamine-oxaloacetate aminotransferase (89.7--209.3 nmol/min . mg protein), glycine oxaloacetate aminotransferase (135--227 nmol/min . mg protein), alanine glyoxylate aminotransferase (66.3--163.2 nmol/min . mg protein) and serine-glyoxylate aminotransferase (57.1--68.4 nmol/min . mg protein). Short term labelling experiments using 14C-glyoxylate show that glycine plays an important role in amino nitrogen transfer in R. acidophila and that the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate were present in cell-free extracts. These data confirm that R. acidophila can satisfy all its' nitrogen requirements by transamination.

Regulation of Enzymes Associated with C-1 Metabolism in Three Facultative Methylotrophs

The levels of the oxidation enzyme methanol dehydrogenase and the serine pathway enzymes, hydroxypyruvate reductase, glycerate kinase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, phosphoenolpyruvate carboxylase, and malyl-coenzyme A lyase, were studied in cells of the facultative methylotrophs Pseudomonas AM1, Pseudomonas 3A2 and Hyphomicrobium X grown on different substrates. Induction and dilution curves for these enzymes suggest they may be regulated coordinately in Hyphomicrobium X, but not in Pseudomonas AM1 or 3A2. Glyoxylate stimulated the serine transhydroxymethylase activity in methanol-grown cells of all three organisms. A secondary alcohol dehydrogenase activity was detected at low levels in Pseudomonas AM1 and Hyphomicrobium X, but not in Pseudomonas 3A2.

Photorespiration mutants of Arabidopsis thaliana deficient in serine-glyoxylate aminotransferase activity

Three mutants of the crucifer Arabidopsis thaliana (Linnaeus) Heynhold were isolated that are completely lacking in activity catalyzed by serine-glyoxylate aminotransferase (EC 2.6.1.45), a peroxisomal enzyme involved in photorespiratory carbon metabolism. These mutants were viable and exhibited normal photosynthesis under conditions that suppressed photorespiration, but they were inviable and photosynthesized at greatly reduced rates under conditions that promoted photorespiration. Serine and glycine accumulated as end products of photosynthesis in the mutants, mostly at the expense of starch and sucrose. The mutants are allelic, and the segregation patterns of plant viability, photosynthetic activity, and enzyme activity in the F(1) and F(2) generations indicated that all the observed effects were caused by a single recessive nuclear mutation. This conclusion was confirmed by the isolation of seven revertants in which viability, photosynthetic capacity, and enzyme activity were simultaneously restored. Mutants of the type described here, in which photorespiration is changed from a merely wasteful process into one that is lethal, may permit the direct selection of secondary mutations that reduce photorespiration.

The Isolation of Pleiotropic Mutants of Pseudomonas aminovorans Deficient in the Ability to Grow on Methylamine and an Examination of their Enzymic Constitution

Summary: Mutants deficient in several enzymes of methylamine oxidation and assimilation have been obtained by treating Pseudomonas aminovorans with ultraviolet light. One of these mutants was studied in detail and was shown to lack the enzymes of trimethylamine and dimethylamine oxidation. In addition, activities of the following enzymes, all postulated as being involved in C1 metabolism, were lost: hydroxypyruvate reductase, serine-glyoxylate aminotransferase, isocitrate lyase, phosphoeno/pyruvate carboxylase, N-methylglutamate dehydrogenase, γ-glutamylmethylamide synthetase, formate dehydrogenase and dye-linked formaldehyde dehydrogenase. In contrast, three enzymes - NAD+,glutathione-linked formaldehyde dehydrogenase, N-methylalanine dehydrogenase and N-methylglutamate synthase - were retained. This phenotype suggests either a lesion in a regulatory gene, a deletion in an early structural gene of an operon or the loss of a plasmid. Results obtained indicated that P. aminovorans possesses an isocitrate lyase-serine pathway.

Cell organelles from crassulacean-acid-metabolism (CAM) plants

Cell organelles were isolated from the CAM plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen by isopycnic centrifugation in sucrose gradients. The inclusion of 2.5% Ficoll in the grinding medium proved to be essential for a satisfactory separation of cell organelles during the subsequent centrifugation. Peroxisomes, mitochondria, and whole and broken chloroplasts were at least partially resolved as judged by marker-enzyme-activity profiles. The isolated peroxisomes contained activities of glycollate oxidase, catalase, hydroxypyruvate reductase, glycine aminotransferase, serine-glyoxylate aminotransferase, and aspartate aminotransferase, comparable to activities found in spinach (Spinacia oleracea L.) leaf peroxisomes. In contrast to spinach, however, only little, if any, particulate malate dehydrogenase activity could be attributed to isolated peroxisomes of the three CAM plants.

Enzyme Regulation in Methylobacterium organophilum

Several enzymes have been assayed in Methylobacterium organophilum grown on different substrates. The enzymes which are involved in growth on C1 compounds were induced by methanol and not repressed by succinate. When succinate-grown bacteria were resuspended in medium containing methanol, four enzymes unique to growth on C1 compounds (hydroxypyruvate reductase, serine-glyoxylate aminotransferase, methanol dehydrogenase and glycerate kinase) were fully induced by the time growth began. When methanol-grown bacteria were resuspended in medium containing succinate, all four enzyme activities decreased. Several mutants unable to grow on C1 compounds were examined for deficiencies in the enzymes specific for growth on these compounds. Seven of the mutants were pleiotropic, and six were not revertible by chemical mutagens, suggesting the possibility of genetic linkage or the presence of a regulon for the genes involved in C1 metabolism.

Isolation of Intact Chloroplasts and Other Cell Organelles from Spinach Leaf Protoplasts

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.

The localization of glycollate-pathway enzymes in Euglena.

Isolation of organelles from broken-cell suspensions of phototrophically grown Euglena gracilis Klebs was achieved by isopycnic centrifugation on sucrose gradients. 2. Equilibrium densities of 1.23g/cm3 for peroxisome-like particles, 1.22g/cm3 for mitochondria and 1.17g/cm3 for chloroplasts were recorded. 3. The enzymes glycollate dehydrogenase, glutamate-glyoxylate aminotransferase, serineglyoxylate aminotransferase, aspartate-alpha-oxoglutarate aminotransferase, hydroxy pyruvate reductase and malate dehydrogenase were present in peroxisome-like particles. 4. Unlike higher plants glycollate dehydrogenase and glutamate-glyoxylate aminotransferase were present in the mitochondria of Euglena. 5. Rates of glycollate and D-lactate oxidation were additive in the mitochondria, and, although glycollate dehydrogenase was inhibited by cyanide, D-lactate dehydrogenase activity was unaffected. 6. Glycollate oxidation was linked to O2 uptake in mitochondria but not in peroxisome-like particles. This glycollate-dependent O2 uptake was inhibited by antimycin A or cyanide. 7. The physiological significance of glycollate metabolism in Euglena mitochondria is discussed, with special reference to its role in photorespiration in algae.

Serine-Glyoxylate aminotransferase from kidney bean ( Phaseolus vulgaris) : II. The reverse reactions

1. 1.|The serine-glyoxylate aminotransferase (EC 2.6.1.45) isolated from Phaseolus vulgaris, which catalyzes aminotransferase reactions between serine-glyoxylate and serine-pyruvate, was shown to catalyze the reverse reactions hydroxypyruvate-glycine and hydroxypyruvate-alanine. 2. 2.|The equilibrium constant for the reaction of serine and glyoxylate to form hydroxypyruvate and glycine was between 32 and 119. The K m values for hydroxypyruvate and glycine were 1.1·10 −3 and 1.1·10 −2 M, respectively. The equilibrium constant for the reaction of serine and pyruvate to form hydroxypyruvate and alanine was 43. The K m values for hydroxypyruvate and alanine were 6.3·10 −4 and 2·10 −2 M, respectively. 3. 3.|The reverse reactions were inhibited by low concentrations of hydroxylamine, but were less sensitive to N-ethylmaleimide and p-hydroxymercuribenzoate. The reverse activities were not specifically affected by NH 4 +.