Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy (Oryza sativa L.)

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

Further characterization of the saccharide specificity of peanut (Arachis hypogaea) agglutinin

2-Dansylamino-2-deoxy-D-galactose (GalNDns) has been shown to bind to peanut (Arachis hypogaea) agglutinin (PNA) in a saccharide-specific manner. This binding was accompanied by a five-fold increase in the fluorescence of GalNDns. The interaction was characterized by an association constant of 0.15 mM at 15° and ΔH and ΔS values of -57.04 kJ·mol-1 and -118.1 J·mol-1.K-1, respectively. Binding of a variety of other mono-, di- and oligo-saccharides to PNA, studied by monitoring their ability to dissociate the PNA-GalNDns complex, revealed that PNA interacts with several T-antigen-related structures, such as β-d-Galp-(1→3)-D-GalNAc, β-D-Galp-(1→3)-α-D-GalpNAcOMe, and β-D-Galp-(1→3)-α-D-GalpNAc(1→3)-Ser, as well as the asialo-G(M1) tetrasaccharide, with comparable affinity, thus showing that this lectin does not discriminate between saccharides in which the penultimate sugar of the β-D-Galp-(1→3)-D-GalNAc unit is the α or β anomer, in contrast to jacalin (Artocarpus integrifolia agglutinin), another anti T-lectin which preferentially binds to β-D-Galp-(1→3)-α-D-GalNAc and does not recognize β-D-Galp-(1→3)-β-D-GalNAc or the related asialo-G(M1) oligosaccharide. These studies also indicated that, in the extended combining region of PNA which accommodates a disaccharide, the primary subsite (subsite A) is highly specific for D-galactose, whereas the secondary subsite (subsite B) is less specific and can accommodate various structures, such as D-galactose, 2-acetamido-2-deoxy-D-galactose, D-glucose, and 2-acetamido-2-deoxy-D-glucose.

Insight into the early stages of thermal unfolding of peanut agglutinin by molecular dynamics simulations

Peanut agglutinin is a homotetrameric nonglycosylated protein. The protein has a unique open quaternary structure. Molecular dynamics simulations have been employed follow the atomistic details of its unfolding at different temperatures. The early events of the deoligomerization of the protein have been elucidated in the present study. Simulation trajectories of the monomer as well as those of the tetramer have been compared and the tetramer is found to be substantially more stable than its monomeric counterpart. The tetramer shows retention of most of its.. secondary structure but considerable loss of the tertiary structure at high temperature. e generation of a This observation impies the molten globule-like intermediate in the later stages of deoligomerization. The quaternary structure of the protein has weakened to a large extent, but none of the subunits are separated. In addition, the importance of the metal-binding to the stability of the protein structure has also been investigated. Binding of the metal ions not only enhances the local stability of the metal-ion binding loop, but also imparts a global stability to the overall structure. The dynamics of different interfaces vary significantly as probed through interface clusters. The differences are substantially enhanced at higher temperatures. The dynamics and the stability of the interfaces have been captured mainly by cluster analysis, which has provided detailed information on the thermal deoligomerization of the protein.

Thermodynamic analysis of three state denaturation of Peanut Agglutinin

Peanut Agglutinin (PNA) is a homotetrameric protein with a very unusual open quaternary structure. During denaturation, it first dissociates into a molten globule like state, which subsequently undergoes complete denaturation. Urea denaturation of PNA at neutral pH has been studied by intrinsic fluorescence spectroscopy and has been fitted to a three state model, A(4) double left right arrow 4I double left right arrow 4U, to get all the relevant thermodynamic parameters. Urea denaturation leads to continuous red shift of wavelength maxima. The molten globule like state is formed in a short range of urea concentration. Refolding of the denatured PNA has been attempted by intrinsic fluorescence study. Refolding by instantaneous dilution shows the occurrence of the formation of an intermediate at a relatively rapid rate, within few seconds. The transition from PNA tetramer to molten globule like state is found to have a Delta G value of similar to 33 kcal/mole while it is similar to 8 kcal/mole for the transition from molten globule like state to a completely denatured state. This in turn indicates that the tetramerization in PNA contributes significantly to the stability of the oligomer.

Molten globule-like state of peanut lectin monomer retains its carbohydrate specificity - Implications in protein folding and legume lectin oligomerization

A central question in biological chemistry is the minimal structural requirement of a protein that would determine its specificity and activity, the underlying basis being the importance of the entire structural element of a protein with regards to its activity vis a vis the overall integrity and stability of the protein. Although there are many reports on the characterization of protein folding/ unfolding intermediates, with considerable secondary structural elements but substantial loss of tertiary structure, none of them have been reported to show any activity toward their respective ligands. This may be a result of the conditions under which such intermediates have been isolated or due to the importance of specific structural elements for the activity. In this paper we report such an intermediate in the unfolding of peanut agglutinin that seems to retain, to a considerable degree, its carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and the quaternary association in legume lectins in particular, where precise subunit topology is required for their biologic activities.

Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli

Using the polymerase chain reaction, the coding sequence for peanut agglutinin (PNA) was cloned and expressed in Escherichia coli. Amplified PNA is identical to previously reported cDNA, suggesting the absence of any introns in PNA gene. Recombinant (re-) PNA forms inclusion bodies in E. coli. Production of PNA was confirmed by probing Western blots with polyclonal anti-PNA immunoglobulin G. Inclusion bodies were solubilized with 6 M guanidine-HCl and renatured by rapid dilution in the presence of metal ions. The renatured lectin was then purified by affinity chromatography. The re-lectin shows carbohydrate-binding properties similar to the natural PNA. This expression system provides a model for future mutagenesis studies of the carbohydrate-binding site and thus facilitates ongoing efforts to explore the molecular basis for the specificity of lectin-carbohydrate interaction.

Crystal structure of peanut lectin, a protein with an unusual quaternary structure

The x-ray crystal structure of the tetrameric T-antigen-binding lectin from peanut, M(r) 110,000, has been determined by using the multiple isomorphous replacement method and refined to an R value of 0.218 for 22,155 reflections within the 10- to 2.95-A resolution range. Each subunit has essentially the same characteristic tertiary fold that is found in other legume lectins. The structure, however, exhibits an unusual quaternary arrangement of subunits. Unlike other well-characterized tetrameric proteins with identical subunits, peanut lectin has neither 222 (D2) nor fourfold (C4) symmetry. A noncrystallographic twofold axis relates two halves of the molecule. The two monomers in each half are related by a local twofold axis. The mutual disposition of the axes is such that they do not lead to a closed point group. Furthermore, the structure of peanut lectin demonstrates that differences in subunit arrangement in legume lectins could be due to factors intrinsic to the protein molecule and, contrary to earlier suggestions, are not necessarily caused by interactions involving covalently linked sugar. The structure provides a useful framework for exploring the structural basis and the functional implications of the variability in the subunit arrangement in legume lectins despite all of them having nearly the same subunit structure, and also for investigating the general problem of "open" quaternary assembly in oligomeric proteins.

Differential binding of peanut agglutinin with lipopolysaccharide of homologous and heterologous Rhizobium

To establish the crucial role of lipopolysaccharide in the initial recognition event of symbiotic peanut-Rhizobium system the ability of various surface polysaccharides isolated from Bradyrhizobium arachis to inhibit the precipitin reaction between peanut agglutinin and asialoganglioside: deoxycholate (1:1) micelles was estimated. It was compared with that of nonsymbiotic systems e.g. Bradyrhizobium japonicum, Bradyrhizobium ciceris and Escherichia coli. Peanut agglutinin was found to interact more strongly with the lipopolysaccharide of Bradyrhizobium arachis than the exopolysaccharide or capsular polysaccharide. The inhibitory capacity of lipopolysaccharides from homologous and heterologous Bradyrhizobium as measured in terms of the concentration necessary for 50 percent inhibition of precipitin reaction were 1428, 500, 410, and 277 times less than that of lactose for Bradyrhizobium arachis, B. japonicum, B. ciceris and Escherichia coli, respectively. These results support that host lectin peanut agglutinin can recognize homologous Bradyrhizobium lipopolysaccharide by virtue of its binding specificity of higher magnitude.

Peanut agglutinin from callus and cell suspension cultures of Arachis hypogaea L

Synthesis of peanut agglutinin was induced in callus and cell suspension cultures of cotyledons of peanut (Arachis hypogaea L.). The lectin was synthesised in cultures through several passages. Biosynthesis of peanut agglutinin was regulated by the type and concentration of exogenous growth regulators and was positively correlated to the growth of the cultures,indicating that the agglutinin may have a role to play during cell growth. Movement of agglutinin from the cells into the medium not only facilitated easy isolation of the lectin but also provided a clue that it may probably serve as a defence molecule. The synthesized lectin purified from culture, was found to be biologically active, and was found to be comparable with the lectin from seeds, in terms of its electrophoretic mobility.

Immune responses to hemagglutinin-neuraminidase protein of peste des petits ruminants virus expressed in transgenic peanut plants in sheep

Peste des petits ruminants (PPR) is an acute, highly contagious disease of small ruminants caused by a morbillivirus, Peste des petits ruminants virus (PPRV). The disease is prevalent in equatorial Africa, the Middle East, and the Indian subcontinent. A live attenuated vaccine is in use in some of the countries and has been shown to provide protection for at least three years against PPR. However, the live attenuated vaccine is not robust in terms of thermotolerance. As a step towards development of a heat stable subunit vaccine, we have expressed a hemagglutinin-neuraminidase (HN) protein of PPRV in peanut plants (Arachis hypogea) in a biologically active form, possessing neuraminidase activity. Importantly. HN protein expressed in peanut plants retained its immunodominant epitopes in their natural conformation. The immunogenicity of the plant derived HN protein was analyzed in sheep upon oral immunization. Virus neutralizing antibody responses were elicited upon oral immunization of sheep in the absence of any mucosal adjuvant. In addition, anti-PPRV-HN specific cell-mediated immune responses were also detected in mucosally immunized sheep. (C) 2010 Elsevier B.V. All rights reserved.

Total peroxidase activity and peroxidase isoforms as modified by salt stress in two cultivars of fox-tail millet with differential salt tolerance

The effect of NaCl on total peroxidase activity, induction of isoperoxidases and lipid peroxidation in 5-day-old seedlings of two contrasting genotypes of Setaria italica L. (Prasad, a salt tolerant cultivar and Lepakshi, a salt susceptible cultivar), was studied. Total peroxidase activity increased under NaCl salinity and the degree of elevation in the activity was salt concentration dependent. Nevertheless, a greater activity was recorded in the tolerant cultivar (cv Prasad) compared to the susceptible (cv Lepakshi) one in all days of sampling. Further, the pattern of isoperoxidases was modified during stress conditions as evident from the electrophoregrams. Although, five acidic isoforms were detected in both cultivars, differences were found between the cultivars. Furthermore, it was observed that acidic isoperoxidases were strongly expressed and an acidic isoperoxidase, A(3p) (27 kDa) is specifically found in the tolerant cultivar (cv Prasad) under NaCl stress. This isoform was partially purified and found to be thermostable with pr 5.5 and the optimum pH 7.4. A close correlation exists between the rate of lipid peroxidation in terms of malonaldehyde (MDA) content and total peroxidase activity per gram fresh weight with salt tolerance of the two cultivars. The tolerant cultivar (cv Prasad) had low MDA content and high total peroxidase activity than the susceptible variety (cv Lepakshi) during salinity stress. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.