Purification and characterization of alanine aminotransferase from Panicum miliaceum leaves

Son, Daeyoung; Jo, Jae Cheol; Sugiyama, Tatsuo

doi:10.1016/0003-9861(91)90470-4

Cited by 33 publications

(35 citation statements)

References 14 publications

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“…The molecular mass of the native enzyme as determined by gel filtration on Superdex-200 was 93.5 kDa, suggesting that the active form of the enzyme exists as a dimer of identical subunits. This is similar to that observed with AlaATs from mesophilic sources (22,34), as well as the aromatic aminotransferases (AroATs) from Thermococcus litoralis (3) and P. furiosus (2), which exist as homodimers. The N-terminal sequence was determined, and the sequence MIRA SKRALSVEYAIR was obtained.…”

Section: Resultssupporting

confidence: 83%

“…A high specificity was found for the transamination of alanine with ␣-ketoglutarate and glutamate with pyruvate (Table 3). This has also been reported for other AlaATs (22,34). However, the enzyme did exhibit significant activity toward aspartic acid and, to a much lesser extent, the branched-chain amino acids with ␣-ketoglutarate.…”

Section: Resultssupporting

confidence: 81%

“…This residue is absent from the Chlamydomonas reinhardtii AlaAT, which has no activity with aspartate (22). The P. furiosus enzyme does, however, have two arginines (Arg 270 and Arg 272) in this region, which could explain the low, but significant, activity with aspartate that is usually not observed with other AlaATs (22,34).…”

Section: Resultsmentioning

confidence: 94%

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Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

et al. 2000

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Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. The k cat /K m values for alanine and pyruvate formation were 41 and 33 s ؊1 mM ؊1 , respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.The hyperthermophilic archaea are a group of phylogenetically related microorganisms which by definition grow optimally at or above 80°C, with a maximal growth temperature of 90°C or higher (5). The majority of these are strictly anaerobic heterotrophs, most of which are obligately dependent upon the reduction of elemental sulfur (S 0 ) to H 2 S. A limited number of facultative S 0 -reducing species are able to grow in the absence of S 0 by means of an alternative fermentative-type metabolism.

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Section: Resultssupporting

confidence: 83%

Section: Resultssupporting

confidence: 81%

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Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

et al. 2000

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“…Bound antibodies were located by linking to alkaline phosphatase-conjugated goat anti-rabbit IgG. The antibodies used for detection, with dilutions in parentheses, were as follows: anti-Nicotiana tabacum Rubisco large subunit (1:10,000; courtesy of Dr. R. Chollet); antiAmaranthus hypochondriacus Rubisco small subunit (1:10,000; courtesy of Dr. J. Berry); anti-Zea mays PPDK (1:50,000; courtesy of Dr. C. Chastain); antiSpinacia oleracea choline monoxygenase (1:1,000; courtesy of Dr. A.D. Hanson); anti-b-subunit of ATP synthase (CF1-b; 1:10,000; courtesy of Dr. A. Barkan); anti-Flaveria bidentis PyT1 (1:10,000; courtesy of Dr. T. Furumoto); antiAmaranthus viridis PEPC (1:100,000; Colombo et al, 1998); anti-histone H3 (1:5,000; Abcam ab1791); serum against the a-subunit of NAD-ME from A. hypochondriacus (1:2,000; Long et al, 1994); anti-SHMT (1:10,000; courtesy of Dr. H. Bauwe); anti-GDC-P (1:10,000; courtesy of Dr. D. Oliver); anti-S. oleracea peroxisomal HPR1 (1:10,000; courtesy of Dr. H. Bauwe; Timm et al, 2008); anti-Panicum miliaceum Ala-AT (1:10,000; specific for C 4 isoform-Ala-AT-2 in the NAD-ME C 4 species P. miliaceum; Son et al, 1991); cAsp-AT (1:10,000; courtesy of Dr. M. Sentoku et al, 2000); and anti-Eleusine coracana mAsp-AT (1:10,000; specific for the mitochondrial isoform in the NAD-ME species E. coracana; Taniguchi and Sugiyama, 1990). The molecular masses of the polypeptides were estimated from a plot of the log of the molecular masses of marker standards versus migration distance.…”

Section: Protein Extraction and Western-blot Analysismentioning

confidence: 99%

Resolving the Compartmentation and Function of C4 Photosynthesis in the Single-Cell C4 Species Bienertia sinuspersici

2011

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Bienertia sinuspersici is a land plant known to perform C 4 photosynthesis through the location of dimorphic chloroplasts in separate cytoplasmic domains within a single photosynthetic cell. A protocol was developed with isolated protoplasts to obtain peripheral chloroplasts (P-CP), a central compartment (CC), and chloroplasts from the CC (C-CP) to study the subcellular localization of photosynthetic functions. Analyses of these preparations established intracellular compartmentation of processes to support a NAD-malic enzyme (ME)-type C 4 cycle. Western-blot analyses indicated that the CC has Rubisco from the C 3 cycle, the C 4 decarboxylase NAD-ME, a mitochondrial isoform of aspartate aminotransferase, and photorespiratory markers, while the C-CP and P-CP have high levels of Rubisco and pyruvate, Pidikinase, respectively. Other enzymes for supporting a NAD-ME cycle via an aspartate-alanine shuttle, carbonic anhydrase, phosophoenolpyruvate carboxylase, alanine, and an isoform of aspartate aminotransferase are localized in the cytosol. Functional characterization by photosynthetic oxygen evolution revealed that only the C-CP have a fully operational C 3 cycle, while both chloroplast types have the capacity to photoreduce 3-phosphoglycerate. The P-CP were enriched in a putative pyruvate transporter and showed light-dependent conversion of pyruvate to phosphoenolpyruvate. There is a larger investment in chloroplasts in the central domain than in the peripheral domain (6-fold more chloroplasts and 4-fold more chlorophyll). The implications of this uneven distribution for the energetics of the C 4 and C 3 cycles are discussed. The results indicate that peripheral and central compartment chloroplasts in the single-cell C 4 species B. sinuspersici function analogous to mesophyll and bundle sheath chloroplasts of Kranz-type C 4 species.Rubisco is the central CO 2 fixation enzyme in plants. It is a bifunctional enzyme, catalyzing the carboxylation as well as the oxygenation of ribulose-bisphosphate (RuBP) to 3-phosphoglycerate (3-PGA) and phosphoglycolate, respectively. While 3-PGA is subsequently utilized in the C 3 cycle to produce triose-phosphates, phosphoglycolate has no known metabolic use (Ogren, 1984) and needs to be detoxified by the plant. This process is known as photorespiration, which is not only energy consuming but also results in a net loss of previously fixed carbon and nitrogen. The rate of carboxylation versus oxygenation through Rubisco is directly related to the availability of CO 2 and the concentration of oxygen competing for the catalytic site of the enzyme. Since Rubisco is thought to have evolved under much higher levels of CO 2 than current atmospheric concentrations, it is believed that photorespiration was insignificant earlier in photosynthetic organisms. Under current atmospheric CO 2 levels, however, significant amounts (e.g. up to 25%) of previously fixed carbon may be lost through RuBP oxygenase activity under conditions that favor photorespiration, like high temperatures or water stres...

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“…Bound antibodies were located by linking to alkaline phosphatase-conjugated goat anti-rabbit IgG or anti-mouse IgG (for actin and tubulin) according to the manufacturer's instructions (Bio-Rad). The antibodies used for detection, with dilutions in parentheses, were as follows: antiAmaranthus viridis PEPC (1:200; Colombo et al, 1998), serum against the a-subunit of NAD-ME from Amaranthus hypochondriacus (1:5,000; Long et al, 1994), anti-Zea mays PPDK (1:10,000; courtesy of Dr. T. Sugiyama), anti-Panicum mileaceum ALA-AT (specific for C 4 isoform-ALAAT-2 in the NAD-ME C 4 species P. milaceum; Son et al, 1991), cASP-AT (1:3,000; courtesy of Dr. M. Sentoku et al, 2000), anti-Eleusine coracana mASP-AT (1:2,500; specific for the mitochondrial isoform in the NAD-ME species E. coracana; Taniguchi and Sugiyama, 1990), anti-SHMT (1:1,000; courtesy of Dr. H. Bauwe), anti-GDC-P (1:1,000; courtesy of Dr. D. Oliver), anti-spinach (Spinacia oleracea) Rubisco LSU (1:10,000; courtesy of Dr. B. McFadden), anti-A. hypochondriacus Rubisco SSU (1:5,000; courtesy of Dr. J. Berry), anti-spinach large subunit of AGPase (1:1,000; courtesy of Dr. T. Okita), anti-chicken gizzard skeletal actin (1:3,000; clone C4; ICN Biomedicals), and anti-bovine b-tubulin (1:3,000; T4026; Sigma-Aldrich).…”

Section: Gel Electrophoresismentioning

confidence: 99%

Leaf Development in the Single-Cell C₄ System in Bienertia sinuspersici: Expression of Genes and Peptide Levels for C₄ Metabolism in Relation to Chlorenchyma Structure under Different Light Conditions

et al. 2008

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Bienertia sinuspersici performs C 4 photosynthesis in individual chlorenchyma cells by the development of two cytoplasmic domains (peripheral and central) with dimorphic chloroplasts, an arrangement that spatially separates the fixation of atmospheric CO 2 into C 4 acids and the donation of CO 2 from C 4 acids to Rubisco in the C 3 cycle. In association with the formation of these cytoplasmic domains during leaf maturation, developmental stages were analyzed for the expression of a number of photosynthetic genes, including Rubisco small and large subunits and key enzymes of the C 4 cycle. Early in development, Rubisco subunits and Gly decarboxylase and Ser hydroxymethyltransferase of the glycolate pathway accumulated more rapidly than enzymes associated with the C 4 cycle. The levels of pyruvate,Pi dikinase and phosphoenolpyruvate carboxylase were especially low until spatial cytoplasmic domains developed and leaves reached maturity, indicating a developmental transition toward C 4 photosynthesis. In most cases, there was a correlation between the accumulation of mRNA transcripts and the respective peptides, indicating at least partial control of the development of photosynthesis at the transcriptional level. During growth under moderate light, when branches containing mature leaves were enclosed in darkness for 1 month, spatial domains were maintained and there was high retention of a number of photosynthetic peptides, including Rubisco subunits and pyruvate,Pi dikinase, despite a reduction in transcript levels. When plants were transferred from moderate to low light conditions for 1 month, there was a striking shift of the central cytoplasmic compartment toward the periphery of chlorenchyma cells; the mature leaves showed strong acclimation with a shade-type photosynthetic response to light while retaining C 4 features indicative of low photorespiration. These results indicate a progressive development of C 4 photosynthesis with differences in the control mechanisms for the expression of photosynthetic genes and peptide synthesis during leaf maturation and in response to light conditions.

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Purification and characterization of alanine aminotransferase from Panicum miliaceum leaves

Cited by 33 publications

References 14 publications

Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

Resolving the Compartmentation and Function of C4 Photosynthesis in the Single-Cell C4 Species Bienertia sinuspersici

Leaf Development in the Single-Cell C₄ System in Bienertia sinuspersici: Expression of Genes and Peptide Levels for C₄ Metabolism in Relation to Chlorenchyma Structure under Different Light Conditions

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Purification and characterization of alanine aminotransferase from Panicum miliaceum leaves

Cited by 33 publications

References 14 publications

Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

Resolving the Compartmentation and Function of C4 Photosynthesis in the Single-Cell C4 Species Bienertia sinuspersici

Leaf Development in the Single-Cell C4 System in Bienertia sinuspersici: Expression of Genes and Peptide Levels for C4 Metabolism in Relation to Chlorenchyma Structure under Different Light Conditions

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Leaf Development in the Single-Cell C₄ System in Bienertia sinuspersici: Expression of Genes and Peptide Levels for C₄ Metabolism in Relation to Chlorenchyma Structure under Different Light Conditions