Hypoxically inducible barley alanine aminotransferase: cDNA cloning and expression analysis

A 1.75 kb cDNA containing the entire coding sequence of the hypoxically inducible alanine aminotransferase (AlaAT) from barley roots was isolated and sequenced. This clone has an open reading frame of 1446 bp, and a deduced amino acid sequence of 482 residues, giving an estimated protein molecular mass of 52 885 Da. RNA blot analysis of barley root tissue showed a 4-fold increase of a single AlaAT-2 mRNA band after 12–24 hours of hypoxic stress, followed by a decrease in message levels after 48 h of hypoxic conditions. AlaAT-2 protein concentration increased in a similar pattern to AlaAT activity in root tissue, to almost 6-fold the aerobic level after 96 h of hypoxic stress. AlaAT-2 activity increased more than 2-fold in roots of Panicum miliaceum exposed to hypoxia, and is the same isoform as the light inducible AlaAT in P. miliaceum leaves. The unique expression patterns of AlaAT-2 in root and leaf tissue upon exposure to different environmental stimuli is also discussed.     

Molecular cloning of an alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum

We have determined the nucleotide sequence of a cDNA encoding AlaAT-2, which is believed to function in the C4-pathway of Panicum miliaceum. An open reading frame (1446 bp) encodes a protein of 482 amino acid residues. The deduced amino acid sequence of AlaAT-2 shows 44.2 and 44.8% homology with the amino acid sequences of AlaATs from rat and human livers, respectively. Northern blot analysis showed that the gene encoding AlaAT-2 in mesophyll and bundle sheath cells was the same and transcribed similarly in the cells. The level of translatable mRNA for AlaAT-2 increased dramatically during greening.     

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.     

Thermostable alanine racemase of Bacillus stearothermophilus. Construction and expression of active fragmentary enzyme

Limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475-8480). We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. The mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologically identical to the wild-type enzyme. The mutant enzyme, i.e. the fragmentary alanine racemase, was active in both directions of the racemization of alanine. The maximum velocity (Vmax) was about half that of the wild-type enzyme, and the Km value was about double. Absorption and circular dichroism spectra of the fragmentary enzyme were similar to those of the wild-type enzyme. An attempt was made to separately express in Escherichia coli a single polypeptide corresponding to each domain, but no protein reactive with the antibody against the wild-type alanine racemase was produced. Therefore, it is suggested that the two polypeptide fragments can fold into an active structure only when they are co-translated and that they correspond to structural folding units in the parental polypeptide chain.     

Photosynthetic CO(2) Fixation Products and Activities of Enzymes Related to Photosynthesis in Bermudagrass and Other Plants

After a 5-second exposure of illuminated bermudagrass (Cynodon dactylon L. var. `Coastal') leaves to ¹⁴CO₂, 84% of the incorporated ¹⁴C was recovered as aspartate and malate. After transfer from ¹⁴CO₂-air to ¹²CO₂-air under continuous illumination, total radioactivity decreased in aspartate, increased in 3-phosphoglyceric acid and alanine, and remained relatively constant in malate. Carbon atom 1 of alanine was labeled predominantly, which was interpreted to indicate that alanine was derived from 3-phosphoglyceric acid. The activity of phosphoenolpyruvate carboxylase, alkaline pyrophosphatase, adenylate kinase, pyruvate-phosphate dikinase, and malic enzyme in bermudagrass leaf extracts was distinctly higher than those in fescue (Festuca arundinacea Schreb.), a reductive pentose phosphate cycle plant. Assays of malic enzyme activity indicated that the decarboxylation of malate was favored. Both malic enzyme and NADP⁺-specific malic dehydrogenase activity were low in bermudagrass compared to sugarcane (Saccharum officinarum L.). The activities of NAD⁺-specific malic dehydrogenase and acidic pyrophosphatase in leaf extracts were similar among the plant species examined, irrespective of the predominant cycle of photosynthesis. Ribulose-1, 5-diphosphate carboxylase in C₄-dicarboxylic acid cycle plant leaf extracts was about 60%, on a chlorophyll basis, of that in reductive pentose phosphate cycle plants.     

Comparative aspects of aminooxyacetate inhibition of glycine oxidation and aminotransferase activity of pea leaf mitochondria

Aminooxyacetate (AOA) was found to inhibit glycine oxidation by pea leaf mitochondria at micromolar levels. The inhibition resulted from an inhibition of both glycine decarboxylase and serine hydroxymethyltransferase (SHMT) activity. Aspartate: 2-oxoglutarate aminotransferase (AsAT) and alanine: 2-oxoglutarate aminotransferase activities of pea leaf mitochondria were also very sensitive to AOA inhibition. Inhibition of both glycine oxidation and aminotransferase activity was likely competitive with respect to the amino group substrate, but also displayed a time-dependent increase in inhibition at constant AOA concentration. In the case of glycine oxidation, this time-dependent component may be related to the rate of penetration of AOA across the inner mitochondrial membrane. Furthermore, the AOA-inhibition of glycine oxidation could be reversed by pyridoxal 5-phosphate (PLP), whereas AOA-inhibited aminotransferase activity was not reversed. The results indicate that the pyridoxal 5-phosphate antagonist, AOA, results in varying types of inhibition depending on the type of enzyme involved.     

Purification and characterization of peroxisomal apo and holo alanine:glyoxylate aminotransferase from bird liver.

Alanine:glyoxylate aminotransferase has been reported to be present as the apo enzyme in the peroxisomes and as the holo enzyme in the mitochondria in chick (white leghorn) embryonic liver. However, surprisingly, birds were found to be classified into two groups on the basis of intraperoxisomal forms of liver alanine:glyoxylate aminotransferase. In the peroxisomes, the enzyme was present as the holo form in group 1 (pigeon, sparrow, Java sparrow, Australian budgerigar, canary, goose, and duck), and as the apo form in group 2 (white leghorn, bantam, pheasant, and Japanese mannikin). In the mitochondria, the enzyme was present as the holo form in both groups. The peroxisomal holo enzyme was purified from pigeon liver, and the peroxisomal apo enzyme from chicken (white leghorn) liver. The pigeon holo enzyme was composed of two identical subunits with a molecular weight of about 45,000, whereas the chicken apo enzyme was a single peptide with the same molecular weight as the subunit of the pigeon enzyme. The peroxisomal holo enzyme of pigeon liver was not immunologically cross-reactive with the peroxisomal apo enzyme of chicken liver, the mitochondrial holo enzymes from pigeon and chicken liver, and mammalian alanine:glyoxylate aminotransferases 1 and 2. The mitochondrial holo enzymes from both pigeon and chicken liver had molecular weights of about 200,000 with four identical subunits and were cross-reactive with mammalian alanine:glyoxylate aminotransferase 2 but not with mammalian alanine:glyoxylate aminotransferase 1.     

Some properties of mitochondrial and cell sap alanine aminotransferase of the rat heart.

Alanine aminotransferase activity is present in mitochondria and the cell sap fraction of the rat myocardium. As distinct from the cell sap form, mitochondrial alanine aminotransferase was significantly inhibited by chloride ions, maleate and incubation medium temperatures of over 40 degrees C. Activity of the cell sap enzyme was inhibited by phosphate and stimulated by temperatures of over 40 degrees C. The pH optimum for cell sap alanine aminotransferase was in the region of 8, while for the mitochondrial enzyme it had a wider range (pH 7.3-8.2). D,L-penicillamine, and antagonist of vitamin B6, inhibited alanine aminotransferase activity equally in intact and tritonized mitochondria and in the cell sap fraction. The activity of mitochondrial and cell sap alanine aminotransferease rose in correlation to the stage of ontogenesis, the maximum increase being observed in the cell sap fraction 14-20 days after birth. The addition of coenzyme to the incubation medium did not affect the activity of either mitochondrial or cell sap alanine aminotransferase. The results indicate that there are two different alanine aminotransferase enzymes in the rat heart, with different intracellular localizations and probably with different regulative functions.     

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 mol CO₂ g^-1 fresh weight h^-1 and a PEP-carboxylase activity of 660.6 mol CO₂ g^-1 fresh weight h^-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C₄ acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total ¹⁴C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in ¹²CO₂. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C₃ plants.Abbreviations RuDP ribulose-1,5-diphosphate - PEP phosphoenol pyruvate - PGA 3-phosphoglycerate     

Regulation of phosphoenolpyruvate carboxylase from maize leaves by nitrate and alanine.

Nitrate and alanine were found to stimulate partially purified maize leaf phosphoenolpyruvate carboxylase under specific assay conditions. Both metabolites stimulated the enzyme at low pH (7.0-7.5) and low substrate levels (1mM phosphoenolpyruvate). Nitrate was found to have a biphasic effect on the enzyme, stimulating at low concentrations (1mM-3mM), with a decrease in stimulation at higher levels. Nitrate caused inhibition of activity at pH 8.0 and although alanine caused some stimulation in activity at pH 8.0 this was not as marked as at the lower pH levels.     

Identification of six novel allosteric effectors of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase isoforms. Physiological context sets the specificity

The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant.     

LEAF ONTOGENY AS A FACTOR IN SUSCEPTIBILITY TO OZONE: AMINO ACID AND CARBOHYDRATE CHANGES DURING EXPANSION

During development (expansion), the cotton leaf passes through a stage in which it is highly susceptible to ozone. This period of susceptibility occurs after the maximum expansion rate but before complete expansion occurs. The period of maximum susceptibility corresponds to a minimum concentration of soluble sugars and free pool amino acids. Specific amino acids such as aspartate, serine, threonine, glutamate, asparagine, alanine, glycine, valine, isoleucine, leucine, histidine, and tryptophan tended to reach a minimum concentration at about the same time leaves were susceptible to ozone. Ozone exposure during the susceptible period is manifested by visible leaf flecking of the upper surface and a dramatic (up to 2 fold) increase in total free pool amino acids. Most individual amino acids tended to increase except for phenylalanine, alanine, phosphoserine, phosphoethanolamine, and ethanolamine. Soluble protein is decreased but not to the same extent that the free pool amino acids increase. Because there is ample evidence that ozone enters the leaf during nonsusceptible periods as well as susceptible periods, it is postulated that ozone damage results because of the depletion of soluble reserves (carbohydrates and amino acids). Perhaps repair of damage cannot occur.     

Developmental profiles and properties of hepatic peroxisomal apo- and mitochondrial holoalanine:glyoxylate aminotransferase during chick embryogenesis

Alanine:glyoxylate aminotransferase was present as the apoenzyme in the peroxisomes and as the holoenzyme in the mitochondria in chick embryos. The peroxisomal enzyme predominated in the early stage and gradually decreased during embryonic development and disappeared after hatching. In contrast, the mitochondrial enzyme gradually increased and predominated in the later stage of chick embryos. Peroxisomal alanine:glyoxylate aminotransferase in chick embryos was a single peptide with a molecular weight of about 40,000. The enzyme differed from the mitochondrial enzyme in the embryos, and mammalian alanine:glyoxylate aminotransferases 1 (with a molecular weight of about 80,000 with two identical subunits) and 2 (with a molecular weight of about 200,000 with four identical subunits) in molecular weights and immunological properties. Mitochondrial alanine:glyoxylate aminotransferase in chick embryos had an identical molecular weight and immunologically cross-reacted with mammalian mitochondrial alanine:glyoxylate aminotransferase 2. Pyridoxal 5'-phosphate dissociated easily from the peroxisomal enzyme saturated with pyridoxal 5'-phosphate. Hepatic aspartate:2-oxoglutarate aminotransferase and alanine:2-oxoglutarate aminotransferase in chick embryos, and hepatic alanine:glyoxylate aminotransferases in different animal species were all present as the holoenzyme.     

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.     

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue.

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.     

The effects of drought stress on free amino acid accumulation and protein synthesis in Brassica napus

Changes in the concentrations of free amino acids and specific organic acids were analysed during the induction of drought stress in Brassica napus . Most of the amino acids showed a characteristic linear increase with the induction of drought stress in Brassica leaves, increasing an average of 5.9-fold over control levels, followed by a reduction in concentration upon rehydration of the plants. Pyruvate concentrations doubled after 4 days of drought stress whereas 2-oxoglutarate concentrations remained relatively constant. The activities of two of the enzymes involved in amino acid biosynthesis, alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1), were also measured. Neither enzyme showed any increase in activity, except when the plants were rehydrated. This suggests that the increase in both alanine and aspartate levels results from the increase in their precursors pyruvate and glutamate and may not require increased enzyme activity. The effect of drought stress upon changes in protein synthesis was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We found that there was an overall decrease in protein synthesis with the induction of drought stress, followed by a resumption of synthesis upon rehydration. In addition, the synthesis of a number of specific polypeptides was found to decrease upon water loss in the leaves.     

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants

The use of mesophyll protoplast extracts from various C₄ species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C₄ photosynthesis. Conditions regulating the formation of the C₄ acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C₄ species, Eleusineindica, an NAD-malic enzyme C₄ species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C₄ species. Light-dependent induction of CO₂ fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO₂ fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C₄ mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO₂ fixation was not.     

The effects of aestivation on the catalytic and regulatory properties of pyruvate kinase from Helix aspersa.

1. Pyruvate kinase was partially purified from the foot, mantle, and digestive gland of active and aestivating snails. 2. At pH 7.0 the apparent Km values for phosphoenolpyruvate (PEP) were 0.064 mmol/l for the enzyme from foot and 0.071 mmol/l for the enzyme from mantle; those for ADP were 0.35 mmol/l for the foot enzyme and 0.33 mmol/l for the mantle enzyme. 3. Both enzymes were inhibited by alanine, and this could be reversed by fructose 1,6-bisphosphate (FBP), although FBP alone was a weak activator. 4. Decreasing the pH to 6.5 markedly increased the inhibition by alanine and reduced the response to FBP. 5. The enzymes from these tissues of aestivating snails showed a small decrease in their affinity for PEP and a small increase in the effectiveness of alanine as an inhibitor. 6. These changes are indicative of a down-regulation of this enzyme which is consistent with the observations in other species during metabolic depression. 7. In contrast the enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics for PEP with a S0.5 of 1.2 mmol/l, but had a markedly higher affinity for PEP, S0.5 = 0.20 mmol/l during aestivation. This may be indicative of other metabolic changes occurring in the digestive gland.