Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli

The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains two different glutamate synthases whose genes, gltB and glsF (previously known as gltS), have been cloned (F. Navarro et al., 1995, Plant Mol. Biol. 27, 753-767). The glsF gene has been expressed in the glutamate auxotrophic Escherichia coli strain CLR207 RecA, but the corresponding protein does not complement the auxotrophy. The transformed strain showed ferredoxin-dependent glutamate synthase (Fd-GOGAT) activity, demonstrating the capability of E. coli for providing and correctly assembling both the iron-sulfur center and the flavin cofactor of the enzyme. Fd-GOGAT (GlsF) is correctly cleaved at Cys37 to form the mature enzyme in E. coli, as occurs with the large subunit of its own NADPH-GOGAT. The recombinant Fd-GOGAT has been purified to electrophoretic homogeneity, using as the main purification step a ferredoxin-affinity chromatography. The pure enzyme, with a molecular mass of about 180 kDa, shows an absorption spectrum characteristic of iron-sulfur flavoproteins. The analyses of the prosthetic groups indicate that Fd-GOGAT contains only one FMN, but no FAD, and one [3Fe-4S](+,0) cluster per molecule. Oxidation-reduction titration, using absorbance changes of the FMN group in the visible region, gave a midpoint redox potential of -200 +/- 25 mV at pH 7.5. The recombinant enzyme is strictly ferredoxin-dependent and shows apparent K(M) values similar to those of the native Synechocystis protein: 4.5 vs 3.5 microM, 2.2 vs 2.5 mM, and 0.6 vs 0.5 mM for ferredoxin, glutamine, and 2-oxoglutarate, respectively. The addition of the reductant dithionite to the enzyme resulted in the loss of the absorption peak at 436 nm, characteristic of oxidized flavins, which was restored by the anaerobic addition of 2-oxoglutarate, in the presence of glutamine.     

Separation of two forms of glutamate synthase in leaves of tomato (Lycopersicon esculentum)

Two forms of glutamate synthase, one dependent on NAD(P)H, and the other on ferredoxin, have been completely separated by ionic exchange chromatography on DEAE cellulose. The NAD(P)H dependent enzyme was further purified by affinity chromatography with Blue Sepharose, showing Km values of 0.5 mM, 0.3 mM and 1.7 μM for glutamine, 2-oxoglutarate and NADH, respectively. Ferredoxin dependent enzyme was also purified to electrophoretic homogeneity; the Km values were 0.5 mM, 0.2 mM and 0.2 μM for glutamine, 2-oxoglutarate and ferredoxin, respectively. These results support the glutamine synthetase-glutamate synthase pathway for nitrogen assimilation.     

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum.

The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-fixing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30 degrees C it showed a specific activity of 32.9 mumol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (Km = 1.25 mM) and NADPH (Km = 64 microM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [S]0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60 degrees C (Ea = 5.1 kcal/mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.     

Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

NADPH/NADH-dependent cold-labile glutamate dehydrogenase in Azospirillum brasilense. Purification and properties

A cold-labile glutamate dehydrogenase (GDH, EC 1.4.1.3) has been purified to homogeneity from the crude extracts of Azospirillum brasilense. The purified enzyme shows a dual coenzyme specificity, and both the NADPH and NADH-dependent activities are equally cold-sensitive. The enzyme is highly specific for the substrates 2-oxoglutarate and glutamate. Kinetic studies with GDH indicate that the enzyme is primarily designed to catalyse the reductive amination of 2-oxoglutarate. The NADP+-linked activity of GDH showed Km values 2.5 X 10(-4) M and 1.0 X 10(-2) M for 2-oxoglutarate and glutamate respectively. NAD+-linked activity of GDH could be demonstrated only for the amination of 2-oxoglutarate but not for the deamination of glutamate. The Lineweaver-Burk plot with ammonia as substrate for NADPH-dependent activity shows a biphasic curve, indicating two apparent Km values (0.38 mM and 100 mM) for ammonia; the same plot for NADH-dependent activity shows only one apparent Km value (66 mM) for ammonia. The NADPH-dependent activity shows an optimum pH from 8.5 to 8.6 in Tris/HCl buffer, whereas in potassium phosphate buffer the activity shows a plateau from pH 8.4 to 10.0. At high pH (greater than 9.5) amino acids in general strongly inhibit the reductive amination reaction by their competition with 2-oxoglutarate for the binding site on GDH. The native enzyme has a Mr = 285000 +/- 20000 and appears to be composed of six identical subunits of Mr = 48000 +/- 2000. The GDH level in A. brasilense is strongly regulated by the nitrogen source in the growth medium.     

Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii

An enzyme which catalyzes the transamination of L-aspartate with 2-oxoglutarate has been purified 400-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138,000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65,000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5-phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-aspartate. L-Aspartate or pyridoxal 5-phosphate, but not 2-oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L-phenylalanine and L-tyrosine with 2-oxoglutarate, and L-serine, L-alanine and L-glutamine with oxaloacetate. L-Aspartate aminotransferase exhibited hyperbolic kinetics for 2-oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L-aspartate and L-glutamate. Apparent Km values were 0.55 mM for 2-oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L-aspartate and 3.88 mM for L-glutamate. Transamination of L-aspartate in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

A novel L-glutamate oxidase from Streptomyces endus. Purification and properties

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2. The Km value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.     

Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803.

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.     

Purification and properties of glutamate synthase from Thiobacillus thioparus.

Glutamate synthase was purified about 250-fold from Thiobacillus thioparus and was characterized. The molecular weight was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380, and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by iron chelators and thiolbinding agents. The enzyme was specific for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and alpha-ketoglutarate, but L-glutamine was partially replaced by ammonia as the amino donor. The Km values of glutamate synthase for NADPH, alpha-ketoglutarate, and glutamine were 3.0 muM, 50 muM, and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3 and 7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase and glutamate synthase, as well as two glutamate dehydrogenases (NADH and NADPH dependent), were present in this organism. This levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM ammonium sulfate. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. It was concluded that the glutamine pathway is important for ammonia assimilation in this autotrophic bacterium.     

Homocitrate synthase from Penicillium chrysogenum. Localization, purification of the cytosolic isoenzyme, and sensitivity to lysine.

Subcellular fractionation of cell-free extracts obtained by nitrogen cavitation showed that Penicillium chrysogenum Q176 contains a cytosolic as well as a mitochondrial homocitrate synthase activity. The cytosolic isoenzyme was purified about 500-fold, and its kinetic and molecular properties were investigated. Native homocitrate synthase shows a molecular mass of 155 +/- 10 kDa as determined by gel filtration and a pH of 4.9 +/- 0.1 as determined by chromatofocusing. The kinetic behaviour towards 2-oxoglutarate is hyperbolic, with Km = 2.2 mM; with respect to acetyl-CoA the enzyme shows sigmoidal saturation kinetics, with [S]0.5 = 41 microM and h = 2.6. The enzyme was inhibited strongly by L-lysine (Ki = 8 +/- 2 microM; 50% inhibition by 53 microM at 6 mM-2-oxoglutarate), competitively with 2-oxoglutarate, in protamine sulphate-treated and desalted cell-free extracts and in partially purified preparations. The extent of this inhibition was strongly pH-dependent. Both isoenzymes are equally susceptible to inhibition by lysine. The same inhibition pattern is shown by the enzyme from strain D6/1014A, which is a better producer of penicillin than strain Q176.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.     

Attachment of bacteria to model solid surfaces: oligo(ethylene glycol) surfaces inhibit bacterial attachment

Abstract An NADP(H)-specific glutamate dehydrogenase of Haloferax mediterranei has been purified to apparent homogeneity and characterised. The purified enzyme was stabilized by glycerol in absence of salt. Glutamate dehydrogenase from Hf. mediterranei is a hexameric enzyme with a native molecular mass of 320 kDa composed of monomers each with a molecular mass of 55 kDa. At pH 8.5 the enzyme has K _ms of 0.018, 0.34 and 4.2 mM for NADP+, 2-oxoglutarate and ammonium, respectively. Amino acid composition and sequence of the first 16 residues of the N-terminus have been determined.     

Enzymes of nitrogen metabolism in legume nodules. Purification and properties of NADH-dependent glutamate synthase from lupin nodules.

An NADH-dependent glutamate synthase has been purified 500-fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, Mr 235000. The optimum pH is 8.5, at which Km values for 2-oxoglutarate, glutamine and NADH are 39 micrometer, 400 micrometer and 1.3 micrometer respectively. The catalytic centre activity is of the order of 70 s-1 and is independent of pH between 6.5 and 9.5. Glutamate synthase is inhibited by glutamic acid, oxaloacetic acid, aspartic acid and asparagine, all competitive with 2-oxoglutarate; and by NAD+, which is competitive with NADH. There is evidence of two flavine prosthetic groups per enzyme molecule.     

Purification and some properties of mitochondrial glutamate:glyoxylate aminotransferase and mechanism of its involvement in glycolate pathway in Euglena gracilis z

Euglena contains glutamate:glyoxylate aminotransferase (GGT) both in mitochondria and in cytosol. Both isoforms were separated from each other by DEAE-cellulose chromatography. The mitochondrial enzyme had an apparent Km of 1.9 mM for glutamate and the cytosolic enzyme 52.6 mM. Mitochondrial GGT was further purified by ammonium sulfate fractionation, isoelectric focusing, and gel chromatography. It had a molecular weight of 141,000 and an isoelectric point of pH 4.88; the optimum pH was 8.5. Its apparent Km values for glutamate and for glyoxylate were 2.0 and 0.25 mM, respectively. In addition to glutamate, mitochondrial GGT used 5-hydroxytryptophan, tryptophan, and cysteine as amino donors in the transamination to glyoxylate. Alanine did not support the activity. The relative activity of the enzyme for amino acceptors on the transamination from glutamate was 4-hydroxyphenylpyruvate greater than phenylpyruvate greater than glyoxylate greater than hydroxypyruvate. Pyruvate and 2-oxoglutarate were not used in the reaction. Evidence that GGT functions mainly in the irreversible transamination between glutamate and glyoxylate is presented. The functional significance of GGT in the glycolate pathway of Euglena is also discussed.     

Purification and properties of glutamate synthase from Nocardia mediterranei.

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.     

Purification and characterization of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from the archaebacterium Methanococcus voltae

A methylviologen and 8-hydroxy-5-deazaflavin(F420)-reducing hydrogenase was purified over 800-fold to near homogeneity from the archaebacterium Methanococcus voltae with 10 U mg-1 F420-reducing activity. It is the only hydrogenase in this organism. The enzyme showed Km values of 16 microM for F420 and 1.2 mM for methylviologen. A turnover number of 1050 min-1 was calculated for the minimal active unit. The protein tends to aggregate. The molecular mass of the minimal active unit is 105 kDa. Larger molecules of 745 kDa were regularly observed. The enzyme was resolved into subunits with molecular masses of 55 kDa, 45 kDa, 37 kDa and 27 kDa by SDS/polyacrylamide gel electrophoresis. Reversible conversion of an anionic into an uncharged form was observed by DEAE-cellulose chromatography with concomitant changes in substrate specificities. The methylviologen-reducing activity was heat-resistant up to 65 degrees C and was not affected by antiserum raised against the native enzyme, while F420 reduction was inactivated by both treatments. Nickel and selenium contents were determined as 0.6-0.7 mol each, FAD content as 1 mol and iron as 4.5 mol/mol protein (105 kDa), respectively. Electron micrographs taken from the purified enzyme show ring-shaped molecules of 18 nm diameter, which represent the high-molecular-mass species of the enzyme.     

Purification and characterization of alanine aminotransferase from Panicum miliaceum leaves

Three alanine aminotransferases, two minor (AlaAT-1, AlaAT-3) and one major (AlaAT-2), were detected by native gel electrophoresis of leaf extracts from Panicum miliaceum L. AlaAT-2 was purified to homogeneity and a specific polyclonal antibody was raised against it which did not react with the other two forms of the enzyme. The enzyme, with an apparent molecular size of 102 kDa, appeared to be a dimer of a single 50-kDa polypeptide. The enzyme has a relatively broad pH optima with similar curves for the forward and reverse directions, ranging between 6.5 and 7.5. The Km values of this enzyme were 6.67, 0.15, 5.00, and 0.33 mM for alanine, 2-oxoglutarate, glutamate, and pyruvate, respectively. The activity of AlaAT-2 was found to increase markedly during leaf greening in parallel with the increase of immunochemically titrated protein, and it is suggested to function in the C4 photosynthetic cycle.