Rosario Hermoso

High-Yield Expression of Pea Thioredoxin m and Assessment of Its Efficiency in Chloroplast Fructose-1,6-Bisphosphatase Activation

A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient. Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P. Schurmann, K. Maeda, and A. Tsugita ([1981] Eur J Biochem 116: 37–45), who did not find Trx m efficiency in FBPase activation. This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation.

Binding site on pea chloroplast fructose-1,6-bisphosphatase involved in the interaction with thioredoxin

When we compare the primary structures of the six chloroplast fructose-1,6-bisphosphatases (FBPase) so far sequenced, the existence of a poorly conserved fragment in the region just preceding the redox regulatory cysteines cluster can be observed. This region is a good candidate for binding of FBPase to its physiological modulator thioredoxin (Td), as this association shows clear differences between species. Using a cDNA clone for pea chloroplast FBPase as template, we have amplified by PCR a DNA insert coding for a 19 amino acid fragment (149Pro-167Gly), which was expressed in pGEMEX-1 as a fusion protein. This protein strongly interacts with pea Td m, as shown by ELISA and Superose 12 gel filtration, depending on pH of the medium. Preliminary assays have shown inhibition of FBPase activity in the presence of specific IgG against the 19 amino acid insert. Surprisingly the fusion protein enhances the FBPase activation in competitive inhibition experiments carried out with FBPase and Td. These results show the fundamental role played by this domain in FBPase-Td binding, not only as docking point for Td, but also by inducing some structural modification in the Td molecule. Taking as model the structural data recently published for spinach photosynthetic FBPase [29], this sequence from a tertiary and quaternary structural point of view appears available for rearrangement.

Cloning, structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme.

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in λgt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3′-untranslated region coding for an mRNA poly(A)+ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39253 daltons (Da), showing 82–88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb)KpnI-SacI fragment of the λgt11 derivative was subcloned between theKpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates ofEscherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, theE. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5. It also had a higher heat sensitivity, with 42% residual activity after heating for 30 min at 60°C, conditions which preserved the native enzyme in a fully active state. These results show the existence of some difference(s) in the conformation of the two FBPases; this could be a consequence of a different expression of the genomic and cDNA clones, or be due to the need for some factor for the correct assembly of the oligomeric structure of the native chloroplast enzyme.

An immunological method for quantitative determination of photosynthetic fructose-1,6-bisphosphatase in leaf crude extracts

An immunological method for quantitative determination of photosynthetic fructose-1,6-bisphosphatase in crude extracts of leaves is proposed. It is based on the ELISA technique, and offers two modifications. A non-competitive technique has a higher sensitivity and is the right option for samples of low fructose-1,6-bisphosphatase content. However, this method is not sufficiently specific when the total protein is higher than 5 μg/cm3; so, despite its lower sensitivity, in these circumstances a competitive technique is more suitable. Thus photosynthetic fructose-1,6-bisphosphatase can be measured without interferences from the gluconeogenic cytosolic enzyme of the photosynthetic cell or from a non-specific phosphatase present in the chloroplast.

Purification and properties of pea (Pisum sativum L.) thioredoxin f, a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (Mr) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an Mr of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at — 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP+-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa Mr = 6324 M−1 · cm−1). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.

Cloning and sequencing of a pea cDNA fragment coding for thioredoxin m.

Thioredoxins are low molecular mass proteins (about 12 kD) engaged in different redox processes (Holmgren, 1985; Buchanan, 1992). From a functional point of view two types of thioredoxins coexist in the plant kingdom. Chloroplast thioredoxins are concerned with activation of some organellar enzymes through a light-mediated reduction mechanism (Jacquot, 1984; Cseke and Buchanan, 1986), whereas cytosolic thioredoxins appear to be engaged in other redox functions, such as the synthesis of deoxyribonucleotides from the corresponding ribonucleotides. Two types of thioredoxins have been found in the chloroplast: thioredoxin f is especially competent in Fru-1,6-bisphosphatase activation, whereas thioredoxin m is more effective in the activation of NADP+malate dehydrogenase (Cseke and Buchanan, 1986; Buchanan, 1992). However, in spite of the same cellular location, both chloroplast thioredoxins show a clear phylogenetic divergence. The m form is structurally close to prokaryotic thioredoxins, and the f type is more similar to those from mammals and yeasts (Hartman et al., 1990). AI1 the thioredoxins so far sequenced show the active center Cys-X-ProCys (X = Gly or Ala), which fulfills the redox function through the establishment of a Cys-Cys bridge (Holmgren, 1985). The only chloroplast thioredoxins from higher plants so far sequenced are the f types from spinach (Kamo et al., 1989) and pea (Lepiniec et al., 1992) and the m type from spinach (Wedel et al., 1992). We now describe the isolation of a cDNA clone coding for pea thioredoxin m, as well as the nucleotide sequence and the deduced primary structure of this chloroplast thioredoxin (Table I).

In-vivo and in-vitro synthesis of photosynthetic fructose-1,6-bisphosphatase from pea (Pisum sativum L.)

Etiolated pea (Pisum sativum L. cv. Lincoln) seedlings do not show any capability for the biosynthesis of chloroplast fructose-1,6-bisphosphatase (FBPase), but the rate of biosynthesis of the increases with the pre-illumination time. This light-induced FBPase synthesis appears to be regulated at the transcriptional level, the response of young leaves being greater than that of mature ones. In-vivo labelling experiments demonstrated by immunoprecipitation, followed by sodium dodecyl sulfate electrophoresis and fluorography, the presence of a 49-kilodalton (kDa) band which corresponds to the mature FBPase subunit. In-vitro translation experiments with a wheat-germ synthesizing system and polyadenylated mRNA isolated from illuminated young pea seedlings have demonstrated the appearance of a 59-kDa labelled band corresponding to the precursor of the FBPase basic subunit. When intact pea chloroplasts were added to the above in-vitro incubation mixture, a labelled 49-kDa subunit similar to that of the in-vivo experiments appeared in the organelle under illumination. From these results we can conclude that a 10-kDa transit peptide bound to the translated pea FBPase subunit exists in the cytosol; this transit peptide is lost during passage through the chloroplast envelope, leaving the mature subunit inside the organelle.

Antigenic Relationships between Chloroplast and Cytosolic Fructose-1,6-Bisphosphatases

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinants between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.

Binding of photosynthetic fructose-1,6-bisphosphatase to chloroplast membranes

Abstract The binding of stromal fructose-1,6-bisphosphatase (FBPase) activity to thylakoid membranes was investigated in lysates of intact spinach and pea chloroplasts. Binding of spinach FBPase increases with the Mg 2+ concentration of the lysis medium in such a way that 20% of the total enzyme activity becomes associated at 15 mM MgCl 2 . Pea chloroplast FBPase showed a similar interaction, with a 12% linkage to the membranes at 25 MgCl 2 . The apparent K 0.5 -values (Mg 2+ concentration for half-maximum binding) were 1.4 mM (spinach) and 2.2 mM (pea). In the presence of 100 mM KCl or NaCl we have found about 40% of the enzyme activity bound to pea thylakoids; in these cases the apparent K 0.05 -values were 23 mM and 26 mM, respectively. In all cases the double-reciprocal and Hill and Scatchard plots were non-linear, the latter showing a succession of negative and positive cooperativities. Both at 20 mM Mg 2+ or 100 mM K + we found no evidence for any influence of pH on enzyme binding in the range tested (pH 6.5–8.0). From these results we suggest that, under illumination, the negative charges of FBPase and stromal-exposed thylakoid proteins become neutralized, which facilitates the binding of the enzyme to some membranous thioredoxin-like protein. The possible physiological significance of this FBPase-thylakoid membrane interaction is discussed from the point of view of the reductive light-activation of this chloroplast stromal enzyme.

Isoenzymes of lactate dehydrogenase (EC 1.1.1.27) in Dicrocoelium dendriticum and Fasciola hepatica (Trematoda)

The isoenzymes of LDH (EC 1.1.1.27) were studied in Dicrocoelium dendriticum and Fasciola hepatica by horizontal electrophoresis on polyacrylamide gel. Six isoenzymes in D. dendriticum and four in F. hepatica were detected. Densitometric scans demonstrated that the enzyme pattern of LDH in D. dendriticum parasitizing hepatic tissue of Capra hircus was clearly different from the one obtained when the trematode parasitized other hosts such as Bos taurus or Ovis aries.