The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.     

Studying Lords and Ladies,Arum maculatum (L.)

Historical uses of Arum maculatum (L.) are summarized. Field and laboratory investigations show plants with purple and pale spadices are equally attractive to flies of the genus Psychoda; and these are attracted to the scent in the absence of visual stimuli. Over 75% of the flies which enter spikes escape. Temperature changes during spadix maturation, and spathe uncurling in the presence of flower tissue, indol-3-yl acetic acid, and gibberellic acid are investigated in the laboratory. Dilute solutions of both phytohormones cause significant spathe uncurling. Further investigations, suitable for project work in schools and colleges, are suggested.     

Effects of Temperature on Electron Transport in Arum maculatum Mitochondria

The effects of temperature upon the respiratory pathways of Arum maculatum mitochondria have been studied. The alternate oxidase sustained a greater proportion of the total respiration at low temperatures than at higher temperatures. Arrhenius plots of respiratory activities show two discontinuities, one at 14°C and one at 21°C. The lower temperature discontinuity was associated with electron transport from succinate dehydrogenase to the alternative oxidase, enzymes that face the inner side of the membrane while the higher temperature discontinuity was associated with electron transport from the external NADH dehydrogenase to cytochrome c oxidase, which face the outer side of the membrane. Both discontinuities resulted in a decrease in the activation energy for electron transport on one side of the membrane. Arrhenius plots of transmembrane electron transport showed discontinuities at both 14° and 21°C but the upper discontinuity resulted in an increase in the activation energy. Activation energies determined for the respiratory activities show that above 21°C the exogenous NADH-cytochrome pathway and the succinate-alternative oxidase pathway were lower than those for the NADH-alternative pathway or the succinate cytochrome pathway.     

Interaction of purified alternative oxidase from thermogenic Arum maculatum with pyruvate

Plant alternative oxidase (AOX) activity in isolated mitochondria is regulated by carboxylic acids, but reaction and regulatory mechanisms remain unclear. We show that activity of AOX protein purified from thermogenic Arum maculatum spadices is sensitive to pyruvate and glyoxylate but not succinate. Rapid, irreversible AOX inactivation occurs in the absence of pyruvate, whether or not duroquinol oxidation has been initiated, and is insensitive to duroquinone. Our data indicate that pyruvate stabilises an active conformation of AOX, increasing the population of active protein in a manner independent of reducing substrate and product, and are thus consistent with an exclusive effect of pyruvate on the enzyme’s apparent V_max.     

Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria

The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.     

Characterisation of 20-kDa lectin-spermagglutinin from Arum maculatum that prevents Chlamydia pneumoniae infection of L-929 fibroblast cells

A novel lectin from the root of Arum maculatum was isolated by saline extraction and purified by cold ethanol precipitation and subsequent fractionation on Superose 6 column. The lectin named A. maculatum agglutinin is a non-glycosylated protein with 20-kDa molecular mass agglutinating human ejaculated spermatozoa, but not human erythrocytes. The agglutination was blocked in the presence of N-acetylneuraminic acid indicating that the lectin is sialoglycoprotein specific. Chlamydia pneumoniae strain AR-39 showed considerable potential to grow in murine L-929 fibroblast cells. Pretreatment of the cell monolayers with purified lectin reduced the entry and intracellular replication of C. pneumoniae. These results suggest that the isolated lectin prevents attachment by binding to a C. pneumoniae specific sialoglycoprotein receptor expressed on the surface of L-929 fibroblast cells.     

Supramolecular structure of the OXPHOS system in highly thermogenic tissue of Arum maculatum

The protein complexes of the mitochondrial respiratory chain associate in defined ways forming supramolecular structures called respiratory supercomplexes or respirasomes. In plants, additional oxidoreductases participate in respiratory electron transport, e.g. the so-called “alternative NAD(P)H dehydrogenases” or an extra terminal oxidase called “alternative oxidase” (AOX). These additional enzymes were previously reported not to form part of respiratory supercomplexes. However, formation of respiratory supercomplexes might indirectly affect “alternative respiration” because electrons can be channeled within the supercomplexes which reduces access of the alternative enzymes towards their electron donating substrates. Here we report an investigation on the supramolecular organization of the respiratory chain in thermogenic Arum maculatum appendix mitochondria, which are known to have a highly active AOX for heat production. Investigations based on mild membrane solubilization by digitonin and protein separation by blue native PAGE revealed a very special organization of the respiratory chain in A. maculatum, which strikingly differs to the one described for the model plant Arabidopsis thaliana: (i) complex I is not present in monomeric form but exclusively forms part of a I + III₂ supercomplex, (ii) the III₂ + IV and I + III₂ + IV supercomplexes are detectable but of low abundance, (iii) complex II has fewer subunits than in A. thaliana, and (iv) complex IV is mainly present as a monomer in a larger form termed “complex IVa”. Since thermogenic tissue of A. maculatum at the same time has high AOX and I + III₂ supercomplex abundance and activity, negative regulation of the alternative oxidase by supercomplex formation seems not to occur. Functional implications are discussed.     

Pro-inflammatory effect in mice of CvL, a lectin from the marine sponge Cliona varians

CvL, a lectin from the marine sponge Cliona varians agglutinated type A papainized erythrocytes and was strongly inhibited by d-galactose and sucrose. Models of leukocyte migration in vivo were used to study the inflammatory activity of CvL through of mouse paw oedema and peritonitis. Effect of CvL on peritoneal macrophage activation was analysed. Effects of corticoids and NSAIDS drugs were also evaluated on peritonitis stimulated by CvL. Results showed that mouse hind-paw oedema induced by subplantar injections of CvL was dose dependent until 50 microg/cavity. This CvL dose when administered into mouse peritoneal cavities induced maxima cell migration (9283 cells/microL) at 24 h after injection. This effect was preferentially inhibited by incubation of CvL with the carbohydrates d-galactose followed by sucrose. Pre-treatment of mice with 3% thioglycolate increases the peritoneal macrophage population 2.3 times, and enhanced the neutrophil migration after 24 h CvL injection (75.8%, p<0.001) and no significant effect was observed in the presence of fMLP. Finally, pre-treatment of mice with dexamethasone (cytokine antagonist) decreased (65.6%, p<0.001), diclofenac (non-selective NSAID) decreased (34.5%, p<0.001) and Celecoxib (selective NSAID) had no effect on leukocyte migration after submission at peritonitis stimulated by CvL, respectively. Summarizing, data suggest that CvL shows pro-inflammatory activity, inducing neutrophil migration probably by pathway on resident macrophage activation and on chemotaxis mediated by cytokines.     

Purification of the plant alternative oxidase from Arum maculatum: measurement, stability and metal requirement

We have purified plant alternative oxidase (AOX) protein from the spadices of thermogenic Arum maculatum (cuckoo pint) to virtual homogeneity. The obtained enzyme fraction exhibits a high specific activity, consuming on average 32 micromol oxygen min(-1) mg(-1), which is completely stable for at least 6 months when the sample is stored at -70 degrees C. This exceptionally stable AOX activity is inhibited approximately 90% (I(50) approximately 10 microM) by 8-hydroxyquinoline (8-OHQ) and also, although to a lesser extent, by other metal chelators such as o-phenanthroline, alpha,alpha'-dipyridyl and EDTA. When inhibited by 8-OHQ, AOX activity is fully restored upon addition of 1.2 mM ferric iron, but neither ferrous iron nor manganese has any effect, whilst zinc decreases activity even further. Furthermore, we have developed a spectrophotometric assay to measure AOX activity in an accurate manner, which will facilitate future steady state and transient kinetic studies. The reliability of this assay is evidenced by retained stability of AOX protein during the course of the reaction, reproducibility of the measured initial rates, an observed 2:1 duroquinol-oxygen stoichiometry and by the fact that, in absolute terms, the measured rates of duroquinone formation and duroquinol disappearance are identical.     

Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria.

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.     

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria

Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca²⁺ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH.     

Iron-sulphur centres in mitochondria from Arum maculatum spadix with very high rates of cyanide-resistant respiration.

X-band electron-paramagnetic-resonance spectroscopy at 4.2--77K combined with measurements of oxidation-reduction potential was used to identify iron--sulphur centres in Arum maculatum (cuckoo-pint) mitochondria. In the oxidized state a signal with a derivative maximum at g = 2.02 was assigned to succinate dehydrogenase centre S-3. Unreduced particles showed additional signals at g = 2.04 and 1.98 (at 9.2 GHz), which may be due to a spin-spin interaction. In the reduced state a prominent signal at g = 1.93 and 2.02 was resolved into at least three components that could be assigned to centres S-1 and S-2 of succinate dehydrogenase (midpoint potentials -7 and -240 mV respectively at pH 7.2) and a small amount of centre N-1b (e'o= -240 mV) of NADH-ubiquinone reductase. In addition, changes in line shape around -10 mV indicated the presence of a fourth component in this signal. The latter was more readily reduced by NADH than by succinate, suggesting that it might be associated with the external NADH dehydrogenase. The iron-sulphur centres of NADH-ubiquinone reductase were present in an unusually low concentration, indicating that the alternative, non-phosphorylating, NADH dehydrogenase containing a low number of iron-sulphur centres may be responsible for most of the high rate of oxidation of NADH.     

Measurements of starch breakdown as estimates of glycolysis during thermogenesis by the spadix of Arum maculatum L.

The rates of starch breakdown at thermogenesis by clubs of the spadices of Arum maculatum L. were measured in individual clubs still attached to the plants. The values obtained were used as estimates of the rate of glycolysis at thermogenesis. Such rates were shown to exceed the maximum catalytic activities of phosphofructokinase (E.C. 2.7.1.11.), aldolase (E.C. 4.1.2.7.), and glyceraldehydephosphate dehydrogenase (E.C. 1.2.1.12.) in developing clubs. The marked increases in the activities of the above enzymes that occur during the development of the club have been shown to be a prerequisite for the attainment of the high rate of glycolysis found at thermogenesis, and thus to be an example of coarse control of glycolysis.     

Effects of 2,4-dinitrophenol and anoxia on the inorganic-pyrophosphate content of the spadix of Arum maculatum and the root apices of Pisum sativum

This work was done to determine whether the inorganic-pyrophosphate (PPi) content of plant tissues changes when the rate of glycolysis is altered. Treatment of excised clubs of the spadix of Arum maculatum L. and root apices of Pisum sativum L. with 2,4-dinitrophenol increased the rates of respiration but had no detectable effects on PPi contents. When the two tissues were subjected to up to 60 min anoxia, no changes in PPi were detected. Anoxia was shown to lead to a fall in ATP and concomitant rises in ADP and AMP in pea roots. It is argued (i) that variation in the rate of glycolysis was not accompanied by detectable changes in PPi content, (ii) that this observation does not favour the view that pyrophosphate fructose 6-phosphate 1-phosphotransferase mediates appreciable entry into glycolysis, and (iii) that PPi content can be maintained when respiratory-chain phosphorylation is inhibited.Abbreviations FW fresh weight - PFK(PPi) pyrophosphate fructose 6-phosphate 1-phosphotransferase - PPi inorganic pyrophosphate     

Pro-inflammatory effect of TWEAK/Fn14 interaction on human umbilical vein endothelial cells

TWEAK, a member of the TNF family, induces cell death in some tumor cell lines, but also induces proliferation of endothelial cells and angiogenesis. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which may be responsible for the proliferation of endothelial cells and angiogenesis. In this study, we investigated the pro-inflammatory effect of TWEAK on human umbilical vein endothelial cells (HUVEC). We demonstrated that TWEAK could not only induce the proliferation and migration but also upregulate the cell surface expression of adhesion molecules such as ICAM-1 and E-selectin, and induce the secretion of chemokines such as IL-8 and MCP-1 in HUVEC. Moreover, by using an anti-Fn14 mAb that blocks the TWEAK/Fn14 interaction, we demonstrated that Fn14 was constitutively expressed on HUVEC and totally mediated the biological effects of TWEAK on HUVEC. These results indicated that TWEAK could induce pro-inflammatory reactions via Fn14 on HUVEC.