Aspartate aminotransferase from Panicum maximum Jacq. var. trichoglume Eyles, a C4 plant: purification, molecular properties, and preparation of antibody

Extracts of the leaf tissue of Panicum maximum Jacq. var. trichoglume Eyles (a phosphoenolpyruvate carboxykinase type of C4 plant) were examined and at least two isoforms of aspartate aminotransferase (EC 2.6.1.1), with different electrophoretic mobilities, were detected. The predominant isoform was purified to homogeneity from mesophyll cells. The purification procedure included fractionation with ammonium sulfate followed by chromatography on diethylaminoethyl-cellulose, Sephacryl S-300, and hydroxyapatite. The purified enzyme had specific activities of 182 and 165 mumol/min/mg protein, measured in terms of the synthesis of oxaloacetate and aspartate, respectively, at pH 8.0. The enzyme, with an apparent molecular size of 100 kDa, appears to be a dimer of a single polypeptide with a molecular size of 42 kDa. Mono specific polyclonal antibodies were raised against the 42-kDa polypeptide. Only a single stained band was detected in extracts of whole leaves by immunoblot analysis with this antibody after two-dimensional polyacrylamide electrophoresis. Furthermore, no difference in mobility was observed between the enzymes extracted from mesophyll and bundle sheath cells on native polyacrylamide gels. These findings are discussed in relation to the other isoform in the leaves of this species.     

Aspartate aminotransferase from wheat germ: purification and kinetic properties.

A convenient method for the purification of aspartate aminotransferase [L-aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1)] from wheat germ is described. An overall purification of 150 fold was achieved. On polyacrylamide gel electrophoresis at pH 8.9 the purified enzyme revealed two protein bands both provided with enzymatic activity. The holoenzyme is readily resolved on conversion to the aminic form and gel-filtration. The apoenzyme is reactivated by pyridoxal-5-phosphate. Kinetic data indicate that a Ping-Pong mechanism is operative similar to that found for the tyrosine aminotransferase by Litwack and Cleland (1968). Phosphate ion behaves as a competitive inhibitor towards the coenzyme. The relatively low affinity between coenzyme and apoenzyme from wheat germ allowed the determination of the dissociation constants for coenzymes (pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate) and of the inhibition constant for phosphate.     

Isolation and characterization of two genetically distant groups of Acetobacter diazotrophicus from a new host plant Eleusine coracana L.

Acetobacter diazotrophicus strains were isolated from Eleusine coracana , a new host plant cultivated along the coast of Tamil Nadu in India. Using a species-specific oligonucleotide primer and PCR amplification, the presence of this bacterium was demonstrated directly in plant tissues, proving its endophytic nature, and it was absent in non-rhizosphere soils. The isolates were also characterized on the basis of typical morphology, electron microscopy and biochemical tests, including nitrogen-fixing efficiency, to assess their diversity. When RAPD analysis was performed on the isolates, they fell into two distinct genetically related groups when compared with the type strain PA1 5 (ATCC 49037). In view of the importance of E. coracana to this region, associated nitrogen-fixing Acetobacter strains may be agronomically important because they could supply part of the nitrogen that the crop requires.     

Three alpha-amylases from malted finger millet (Ragi,Eleusine coracana,Indaf-15)--purification and partial characterization

Three alpha-amylases (E.C. 3.2.1.1) were purified to apparent homogeneity from 72 h finger millet malt by three step purification via fractional acetone precipitation, DEAE-Sephacel ion exchange and Sephacryl S-200 gel permeation chromatographies with a recovery of 6.5, 2.9, 9.6% and fold purification of 26, 17 and 31, respectively. alpha-Nature of these amylases was identified by their ability to rapidly reduce the viscosity of starch solution and also in liberating oligosaccharides of higher D.P. and were accordingly designated as amylases alpha-1((b)), alpha-2 and alpha-3, respectively. These amylases, having a molecular weight of 45+/-2 kDa were found to be monomeric. The pH and temperature optima of these alpha-amylases were found to be in the range of 5.0-5.5 and 45-50 degrees C, respectively. K(m) values of these amylases for various cereal starches varied between 0.59 and 1.43%. Carbodiimide (50 mM) and metal ions such as Al(3+), Fe(2+), and Hg(2+) (5 mM) have completely inhibited these enzymes at 45 degrees C. Amino acid analysis of these enzymes indicated high amounts of glycine which is an unusual feature of these enzymes.     

Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.)

A carboxylesterase (EC 3.1.1.1) was purified from germinated finger millet by ammonium sulphate fractionation, diethylaminoethyl-cellulose chromatography and Sephadex G-200 filtration. The homogeneity of the enzyme was established by Polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme has a single polypeptide chain with a molecular weight of 70,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to, basic amino acid residues. The isoelectric pH of the enzyme was found to be 5·1. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was more sensitive to organophosphate inhibitors than carbamates. The rate constantsk _i andl ₅₀ for different inhibitors were calculated. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear noncompetitive inhibition with 1-naphthol     

Large-scale preparation,purification, and characterization of hyaluronan oligosaccharides from 4-mers to 52-mers

Hyaluronan (HA) was depolymerized by partial digestion with testicular hyaluronidase and separated into size-uniform HA oligosaccharides from 4-mers to 52-mers by anion exchange chromatography after removal of the hyaluronidase. The purity and size of each HA oligosaccharide was confirmed by using HPLC analyses, FACE, and ESI-MS. (1)H and (13)C NMR assignments and elemental analyses were obtained for each HA oligosaccharide. Endotoxins, proteins, and DNA were absent or in trace amounts in these HA oligosaccharides. Gram/mg-scale hyaluronan oligosaccharides were obtained from 200 g of HA starting material. These pure, size-uniform, and large range of HA oligosaccharides will be available for investigating important biological functions of HA, such as for the determination of the size(s) of HA oligosaccharides that induce angiogenesis or mediate inflammatory responses, and to interact with HA-binding proteins and receptors both in in vitro and in vivo studies.     

Aspartate aminotransferase in alfalfa root nodules : I. Purification and partial characterization

Aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1 [AAT]), a key enzyme in the assimilation of C and N compounds, was purified from the cytosol of alfalfa (Medicago sativa L.) root nodules. Isoforms that increased during nodule development, AAT-2a, AAT-2b, and AAT-2c, were purified greater than 447-fold to apparent homogeneity, and high titer polyclonal antibodies were produced. The native molecular weight of the AAT-2 isoforms was approximately 80 kilodatons with a subunit molecular weight of 40 kilodatons, indicating that the holoenzymes are dimers. The AAT-2 isoforms comprised approximately 0.4% of the total soluble nodule protein. The AAT specific activity was measured in leaf, stem, root, and nodule organs, and zymograms of each were compared. Enzyme activity was 4- to 37-fold greater in effective (nitrogen fixing) nodules than in leaves, stems, and roots. Effective nodule AAT-specific activity was 3- to 8-fold greater than that of plant-controlled ineffective nodules. No differences in K_m were observed between AAT-1 and AAT-2. Antibodies raised against AAT-2 were more selective against AAT-2 than AAT-1. Evidence obtained from zymograms suggests that the expression of alfalfa nodule AAT is controlled at two different gene loci, AAT-1 and AAT-2, resulting in different dimeric isoforms.     

Simultaneous purification and characterization of aspartate aminotransferase isoenzymes from chicken liver

Cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) were purified to homogeneity from chicken liver, without previous fractionation of the subcellular components. The procedure includes initial heat treatment and ammonium sulfate fractionation. The two isoenzymes can then be separated by a DEAE-Sepharose chromatography using a linear gradient of L-aspartate (reaction substrate). The separated fractions can be further purified by a parallel step with HA-Ultrogel prior to octyl-Sepharose (c-AAT) and CM-Sepharose (m-AAT) chromatographies. Michaelis constants, pI values, inhibition by adipate and subforms generation with time were studied for both isoenzymes.     

Aspartate: 2-oxoglutarate aminotransferase from trichomonas vaginalis. Identity of aspartate aminotransferase and aromatic amino acid aminotransferase.

Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.     

Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor

The technique of three-phase partitioning (TPP) was used to purify a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana). This process of purification is a potential method used for separation of proteins directly from large volumes of crude suspension. It involves the addition of a salt (ammonium sulphate) to the crude extract followed by the addition of an organic solvent (t-butanol). The addition of t-butanol, in the presence of ammonium sulphate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The process was carried out in two steps. The various conditions required for attaining efficient purification of the protein fractions were optimized. It was seen that 30% ammonium sulphate saturation with 1:1 ratio of crude extract to tert-butanol gave 8.9- and 8.65-fold purification with 83% and 80% yield of amylase inhibitor and trypsin inhibitor, respectively, in step I. In TPP-step II, 60% ammonium sulphate saturation and ratio of aqueous phase to t-butanol of 1:2 gave maximum 20.1- and 16-fold purification with 39.5% and 32% yield of amylase inhibitor and trypsin inhibitor, respectively. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the inhibitor protein showed substantial purification and the molecular weight of the protein was found to be 14 kDa.     

Molecular cloning,characterization and three-dimensional structure prediction of Lipoxygenase from Finger millet [Eleusine coracana (L.) Gaertn.] germinating seedlings

Lipoxygenases (LOXs) are functionally diverse class of non-heme iron containing dioxygenases. They involve in multiple physiological processes in plants and animals. In current study a full length cDNA of LOX has been cloned from finger millet germinating seedlings using PCR and RACE PCR methods. The ORF (2661 bp) encodes 887 amino acid residues, with an approximate molecular weight of 100.8 kDa and an isoelectric point of 5.90. The sequence exhibited significant similarity to already known LOXs. It comprises of a putative PLAT domain and an iron-binding catalytic domain. Sequence analysis demonstrated that this LOX is most probably a type 9-LOX, and shared highest identity (85 %) with the Setaria italica 9-LOX. TargetP predicted EcLOX as a cytoplasmic protein. It has an optimum pH of about 6.4 and increased lipid peroxidation levels were observed on day 5 of germination. Semi-quantitative RT-PCR analysis showed that EcLOX gene expression was preferentially higher on day 4 after germination, and in growing leaf and root tissues. Its expression could be induced by Methyl Jasmonate (MeJA), but not by Salicylic acid (SA). The 3-dimensional structure of the EcLOX was modeled using in silico approach and Molecular dynamics (MD) simulations were performed. Further, to understand the protein active site, docking studies were performed with substrates and inhibitors. In summary, EcLOX protein was characterized using various molecular, biochemical and in silico methods which improves our understanding of these fascinating enzymes.     

Prebiotic Activity of Purified Xylobiose Obtained from Ragi (Eleusine coracana, Indaf-15) Bran

The role of prebiotics in improving human health has attracted global attention and the research is mostly focused on the strains belonging to the genera Bifidobacterium and Lactobacillus. Non-digestible oligosaccharides hold significant role in recent research due to their prebiotic nature. Soluble polysaccharides (SP, 14.4%), isolated from ragi bran consisted mainly of arabinose and xylose with minor quantities of rhamnose, mannose, galactose and glucose. Ragi bran SP subjected to purified endoxylanase (from 96 h ragi malt) treatment to obtain xylo-oligosaccharides which were further purified on Biogel P-2 followed by HPLC. The purified oligosaccharide yielded (RO-1; 17.9%) was identified as xylobiose by electrospray ionization mass spectrometry (282 + 23 = 305) and ¹HNMR. In vitro studies carried out using Bifidobacterium and Lactobacillus sp. proved the prebiotic nature of the crude xylo-oligosaccharides (XOs) and RO-1. Acetate was found to be the chief short chain fatty acid released during fermentation of both crude XOs and purified xylobiose and 24 h bacterial culture showed high xylanase activity (1020–1690 μU min⁻¹).     

Invertase inhibitor from potatoes: purification, characterization, and reactivity with plant invertases

Invertase inhibitor was extracted from potato tubers and purified nearly 1000-fold. The purification procedure involved precipitation at pH 4.0, fractionation with ammonium sulfate, adsorption on alumina Cγ gel, and gel filtration on Sephadex G-100 and DEAE-Sephadex A-50. The product obtained was homogeneous to electrophoresis on polyacrylamide gel. Exclusion chromatography on Sephadex G-100 indicated a molecular weight of about 17,000. The inhibitor did not inhibit yeast, Neurospora, and several plant invertases. It completely inhibited potato tuber invertase and a number of other plant invertases. Some plant invertases were partially inhibited.     

Structural analysis of arabinoxylans isolated from native and malted finger millet (Eleusine coracana, ragi)

Structural elucidation of purified arabinoxylans isolated from finger millet and its malt by methylation, GLC-MS, periodate oxidation, Smith degradation, NMR, IR, optical rotation, and oligosaccharide analysis indicated that the backbone was a 1,4-beta-D-xylan, with the majority of the residues substituted at C-3. The major oligosaccharide generated by endo xylanase treatment was homogeneous with a molecular weight of 1865 Da corresponding to 14 pentose residues as determined by MALDI-TOF-MS and gel filtration on Biogel P-2. The structural analysis of this oligosaccharide showed that it contained 8 xylose and 6 arabinose residues, substituted at C-3 (monosubstituted) and at both C-2 and C-3 (disubstituted).     

Natural plant enzyme inhibitors. Characterization of an unusual alpha-amylase/trypsin inhibitor from ragi (Eleusine coracana Geartn.).

An inhibitor I-1, capable of acting on both alpha-amylase and trypsin, was purified to homogeneity from ragi (finger-millet) grains. The factor was found to be stable to heat treatment at 100 degrees C for 1 h in the presence of NaCl and also was stable over the wide pH range 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in gradual loss of both the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex was demonstrated by chromatography on a Bio-Gel P-200 column. This indicated that the inhibitor is 'double-headed' in nature. The inhibitor was retained on a trypsin-Sepharose 4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery of amylase-inhibitory and trypsin-inhibitory activities. alpha-Amylase was retained on a trypsin-Sepharose column to which inhibitor I-1 was bound, but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-trinitrobenzenesulphonic acid resulted in complete loss of amylase-inhibitory activity but only 40% loss in antitryptic activity. Modification of arginine residues by cyclohexane-1,2-dione led to 85% loss of antitryptic activity after 5 h, but no effect on amylase-inhibitory activity. The results show that a single bifunctional protein factor is responsible for both amylase-inhibitory and trypsin-inhibitory activities with two different reactive sites.     

Purification and Characterization of NAD Malic Enzyme from Leaves of Eleusine coracana and Panicum dichotomiflorum

NAD malic enzyme (EC 1.1.1.39), which is involved in C₄ photosynthesis, was purified to electrophoretic homogeneity from leaves of Eleusine coracana and to near homogeneity from leaves of Panicum dichotomiflorum. The enzyme from each C₄ species was found to have only one type of subunit by SDS polyacrylamide gel electrophoresis. The M_r of subunits of the enzme from E. coracana and P. dichotommiflorum was 63 and 61 kilodaltons, respectively. The native Mr of the enzyme from each species was determined by gel filtration to be about 500 kilodaltons, indicating that the NAD malic enzyme from C₄ species is an octamer of identical subunits. The purified NAD malic enzyme from each C₄ species showed similar kinetic properties with respect to concentrations of malate and NAD; each had a requirement for Mn²⁺ and activation by fructose- 1,6-bisphosphate (FBP) or CoA. A cooperativity with respect to Mn²⁺ was apparent with both enzymes. The activator (FBP) did not change the Hill value but greatly decreased K_0.5 (the concentration giving half-maximal activity) for Mn²⁺. The enzyme from E. coracana showed a very low level of activity when NADP was used as substrate, but this activity was also stimulated by FBP. Significant differences between the enzymes from E. coracana and P. dichotomiflorum were observed in their responses to the activators and their immunochemical properties. The enzyme from E. coracana was largely dependent on the activators FBP or CoA, regardless of concentration of Mn²⁺. In contrast, the enzyme from P. dichotomiflorum showed significant activity in the absence of the activator, especially at high concentrations of Mn²⁺. Both immunodiffusion and immunoprecipitation, using antiserum raised against the purified NAD malic enzyme from E. coracana, revealed partial antigenic differences between the enzymes from E. coracana and P. dichotomiflorum. The activity of the NAD malic enzyme from Amaranthus edulis, a typical NAD malic enzyme type C₄ dicot, was not inhibited by the antiserum raised against the NAD malic enzyme from E. coracana.     

Purification and characterization of a multienzymatic complex in sugarcane, a C4 plant

We have discovered a multienzymatic complex in fresh young sugarcane leaves. This complex is constituted of three enzymes: PEPcase, NADP-MDH and malic enzyme. After successive molecular sieving chromatography, we have obtained a highly purified sample of the complex which has a molecular weight of 711 kDa. Its functional interest has been evaluated by comparing the kinetic properties of the enzymes in their free forms to those in their complexed form. We show that the association of the three enzymes leads to important changes in their respective kinetic properties.