Antonio Joaquín Santos Márquez

Characterization of Three Functional High-Affinity Ammonium Transporters in Lotus japonicus with Differential Transcriptional Regulation and Spatial Expression

Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{{+}}\) \end{document} is also a regulator of early and late symbiotic interaction steps. Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation. Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus. Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant. Measurement of [14C]methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{{+}}\) \end{document}. The Ki for ammonium was 1.7, 3, and 15 μm for LjAMT1;1, 1;2, and 1;3, respectively. Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 > 1;2 > 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression. Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation. Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO2 concentrations, which also suppress photorespiration. Tissue and cellular localization of LjAMT1 genes expression, using promoter-β-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial localization in different organs, including nodules, suggesting differential roles in the nitrogen metabolism of these organs.

Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme

The NH2-terminal sequences of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) purified from barley (Hordeum vulgare L.) and Chlamydomonas reinhardtii (Dangeard), and of a barley peptide, were determined and the barley sequences were used to design oligonucleotide primers for the polymerase chain reaction. A specific 1.3-kilobase (kb) cDNA fragment specifying the NH2-terminal one-third of the mature barley polypeptide, was amplified, cloned and sequenced. The NH2-terminus of plant Fd-GOGAT is highly conserved and homologous to the NH2-terminus of the heavy subunit of Escherichia coli NADPH-GOGAT. Based on sequence homologies, we tentatively identified the NH2-terminal region of Fd-GOGAT as the glutamine-amidotransferase domain, which is related to the corresponding domain of the purF-type amidotransferases. The Fd-GOGAT cDNA clone, and polyclonal antibodies raised against the barley enzyme, were used to analyse four Fd-GOGAT-deficient photorespiratory mutants. Three mutants (RPr 82/1, RPr 82/9 and RPr 84/82) had no detectable Fd-GOGAT protein in leaves, while the fourth (RPr 84/42) had a small amount of cross-reacting material. Hybridization to Northern blots of total leaf RNA revealed that both RPr 82/9 and RPr 84/82 were indistinguishable from the parental line (Maris Mink), having normal amounts of a 5.7-kb mRNA species. On the other hand, RPr 82/2 and RPr 84/42 each contained two distinct hybridizing RNA species, one of which was larger than 5.7 kb, the other smaller. Using a set of wheat-barley telosomic addition lines we have assigned the Fd-GOGAT structural locus to the short arm of chromosome 2.

Immunological studies of ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from photosynthetic organisms

Polyclonal antiserum specific for ferredoxin-nitrite reductase (EC 1.7.7.1) from the green alga Chlamydomonas reinhardii recognized the nitrite reductase from other green algae, but did not cross-react with the corresponding enzyme from different cyanobacteria or higher plant leaves. An analogous situation was also found for ferredoxin-glutamate synthase (EC 1.4.7.1), using its specific antiserum. Besides, the antibodies raised against C. reinhardii ferredoxin-glutamate synthase were able to inactivate the ferredoxin-dependent activity of nitrite reductase from green algae.These results suggest that there exist similar domains in ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from green algae. In addition, both types of enzymes share common antigenic determinants, probably located at the ferredoxin-binding domain. In spite of their physicochemical resemblances, no apparent antigenic correlation exists between the corresponding enzymes from green algae and those from higher plant leaves or cyanobacteria.

Functional properties of purified ferredoxin-glutamate synthase from Chlamydomon as reinhardtii

Abstract The ferredoxin-glutamate synthase (EC 1.4.7.1) from the green alga Chlamydomonas reinhardtii has been purified to apparent homogeneity (sp. act. 275 nkat/mg) by a fast method which used phenyl-Sepharose chromatography as a basic step. The enzyme molecule contains 1377 amino acid residues with a high proportion of hydrophobic amino acids, particularly proline, as well as glycine and threonine. Fifteen cysteines are also present, 11 of which are as free sulphydryl residues and four form two disulphide bridges. At least two cysteines are essential for enzyme activity and the involvement of sulphhydryl groups in the interaction of the protein with the prosthetic groups or l -glutamine is suggested. The purified enzyme is stimulated by thiols and is also significantly inhibited in vitro by several sulphhydryl reagents as well as l -glycine, l -serine, glycolate and glyoxylate.

Physicochemical Properties of Ferredoxin from Chlamydomonas reinhardii

Abstract Ferredoxin from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity by an easy and fast procedure with a high yield (25 -30 mg/250 g wet weight of cells). An average molecular weight of 11800 was calculated from sedimentation coefficient (1.70 S) and Stokes radius (1.75 nm) data, sodium dodecyl sulfate-electrophoresis, and amino acid composition. Absorption spectrum show ed maxima at 276, 330, 420 and 460 nm in the oxidized form, with an absorption ratio (A420/A276) of 0.54 and an extinction coefficient of 8.38 mᴍ-1· cm -1 at 420 nm. R educed ferredoxin show ed a single peak at 276 nm with shoulders at 284, 310, 390, 469 and 537 nm and at liquid helium temperatures gave EPR signals at g = 1.877, 1.951 and 2.045. The protein has an isoelectric point of 3.30, and one (2Fe-2S)-cluster per molecule with a midpoint potential, at pH 7.5, of -410 mV (n=1) . The m olecule of C. reinhardii ferredoxin consists of 95-99 amino acid residues which includes the full com plement of amino acids, being alanine the most abundant.

Proteolysis of barley (Hordeum vulgare L.) T. ferredoxin-glutamate synthase affects ferredoxin- and methyl viologen-dependent enzyme activities differently

Summary Ferredoxin-glutamate synthase (Fd-GOGAT; E.C. 1.4.7.1) T. from barley undergoes a spontaneous proteolytic cleavage in crude extracts, yielding two fragments of 69 and 96 kDa. The breakdown of Fd-GOGAT polypeptide affects ferredoxin- and methyl viologen dependent activities of the enzyme differently: whereas ferredoxin-dependent activity is severely diminished, methyl viologen-dependent activity is fully retained. Free ferredoxin protects the Fd-GOGAT against proteolytic cleavage and the specific decay of ferredoxin-dependent enzyme activity. Therefore, it is shown for the first time that both enzyme activities can be functionally distinguished in this enzyme. The results are also, in our opinion, of crucial importance in measurements of Fd-GOGAT, given that both reduced ferredoxin and methyl viologen have been used indistinctly as far as electron donors for the Fd-GOGAT reaction are concerned.

Identification of an essential cysteinyl residue for the structure of glutamine synthetase α from Phaseolus vulgaris

We have studied the possible role, in a plant glutamine synthetase (GS), of the different cysteinyl residues present in this enzyme. For this purpose we carried out the site-directed mutagenesis of the cDNA for α-GS polypeptide from Phaseolus vulgaris in the positions corresponding to Cys-92, Cys-159, and Cys-179, followed by heterologous expression in E. coli and enzymatic characterisation of WT and mutant proteins. The results show that neither Cys-92 nor Cys-179 residues were essential for enzyme activity, but the replacement of Cys-159 by alanine or serine strongly affects the quaternary structure and function of the GS enzyme molecule, resulting in a complete loss of enzymatic activity. Other studies using sulfhydryl specific reagents such as pHMB (p-hydroxymercuribenzoate) or DTNB (5,5′-dithiobis-2-nitrobenzoate) confirmed that the profound inhibition produced is associated with an important alteration of the quaternary structure of GS, and suggest that Cys-159 might be the residue responsible for the enzyme inhibition. All these results suggest that the Cys-159 residue is essential for the enzyme structure. The results are also consistent with previous reports based on classical biochemistry studies indicating the presence of essential cysteinyl residues for the enzyme activity of higher plant GS.

Dissociation of FAD from the NAD(P)H-Nitrate Reductase Complex from Ankistrodesmus braunii and Role of Flavin in Catalysis

Ankistrodesmus braunii NAD(P)H-nitrate reductase is a complex hemoflavomolybdoprotein composed by eight similar subunits. The flavin prosthetic group, identified as FAD, is essential for the NAD(P)H-dependent activities of the complex, and is located before the heme chromo- phore in the enzyme electron transport chain from reduced pyridine nucleotides to nitrate. Fluorescence studies indicate that nitrate reductase can dissociate about 80% of its FAD by incubation at room temperature, the flavin dissociation being followed by a parallel decrease of NADH-nitrate reductase activity. Dissociation of FAD from the protein is easily increased by dilution or prolonged dialysis of the enzyme preparations. However, exogenous FAD specifically prevents the dissociation of enzyme-bound flavin, and protects the NAD(P)H-dependent activities. The Km for FAD, as a protector of NADH-cytochrome c reductase activity, is 4 nᴍ. In addition, dithioerythritol also prevents the flavin dissociation, and therefore the presence of free sulphydryl groups in the FAD-domain is suggested. FAD-depleted nitrate reductase, obtained by several methods, is unable to recover its original activity when incubated in the presence of FAD alone or with thiols.

Studies by affinity chromatography on the NAD(P)H and FAD sites of nitrate reductase from ankistrodesmusbraunii

Abstract Both native NAD(P)H-nitrate reductase (E.C. 1.6.6.2.) from Ankistrodesmusbraunii and the FAD-depleted enzyme are adsobed on blue dextran—Sepharose at two different sites. The binding of the native enzyme involves the NAD(P)H active site, while that of the deflavoenzyme uses the FAD site. The holoenzyme can be specifically eluted by 0.5 mM NAD(P)H, but the elution of the deflavoenzyme was achieved only by the simultaneous addition of 1 mM FAD and 0.5 M KCl to the washing buffer. Incubation of native or flavin-free nitrate reductases with p-hydroxymercuribenzoate prevents adsorption of both types of enzyme on blue dextran—Sepharose, which indicates the presence of sulphydryl groups in the sites for NAD(P)H and FAD. Binding studies of the holoenzyme on three different kinds of NAD-Agarose indicate that the NAD(P)H-domain is acting as a crevice in which the nicotinamide ring of the nucleotide should be placed at the bottom. In addition, the structure of the binding site for FAD seems to be similar to that for NAD(P)H.

Mármoles para la Colegiata de Osuna: : la nueva solería y otras actuaciones en su fábrica entre 1770 y 1804

espanolEn este articulo se da a conocer todo el proceso historico que acontecio en la Colegiata de Osuna durante la colocacion de la nueva soleria del templo entre los anos de 1770 y 1804. EnglishThis paper discloses the whole historical process of placing a new flooring at the Colegiata de Osuna between 1770 and 1804.