Cucurbitacin 19-hydroxylase in Cucurbita maxima

The enzymatic hydroxylation of the C-19-methyl group of cucurbitacin B and D was observed in partly purified preparations obtained from the unripe fruit of Cucurbita maxima. Assay methods were developed and the pH optimum, cofactor requirements, and substrate specificity determined.     

Kaurenolide biosynthesis in a cell-free system from Cucurbita maxima seeds

A new product obtained by incubation of [2-¹⁴C ]-mevalonic acid with a cell-free system from Cucurbita maxima endosperm was identified by GC-MS as ent-kaura-6,16-dien-19-oic acid. When this compound was reincubated with the microsomal fraction it was converted to 7β-hydroxykaurenolide and hence to 7β,12α-dihydroxykaurenolide. The dienoic acid was also obtained by incubation of ent-kaurene, ent1-kaurenol, ent-kaurenal and ent-kaurenoic acid, but not ent-7α-hydroxykaurenoic acid, with the microsomal fraction. Thus, in the C. maxima cell-free system, the kaurenolides are formed by a pathway which branches from the GA pathway at ent-kaurenoic acid and proceeds via the dienoic acid.     

Multiple forms of phytase in germinating cotyledons of Cucurbita maxima

Multiple forms of phytase (myoinositol hexaphosphate phosphohydrolase, EC 3.1.3.8) have been isolated in highly purified forms from germinating Cucurbita maxima cotyledons using acetone and ammonium sulphate fractionation, Sephadex gel filtration and ion exchange chromatography on DEAE- and CM-cellulose. Gel filtration produced two peaks of phytase activity; phytase I (high MW) and phytase II (low MW). Phytase I was further resolved into 4 distinct species on CM-cellulose and these were designated phytase IA, IB, IC and ID, according to their elution order. On the other hand, phytase II remained as a single species with a purification of 35-fold. The MWs of each phytase I species were identical (MW 66 500 ± 4000) and they were twice the MW of phytase II (MW 32 400 ± 4000) indicating that I and II may be structurally related. The properties of various molecular forms were compared. The difference in properties between phytase II and phytase I isoenzymes (IA, IB, IC and ID) was more pronounced than that observed among the isoenzymes of phytase I alone.     

Properties of ascorbate oxidase isozymes

The ascorbate oxidase of two squash cultivars was resolved into five molecular forms by gel electrophoresis; that of cucumber was resolved into three forms. Molecular weight estimates by Sephadex gel filtration and interconversions of these forms strongly suggest the presence of a monomeric form of MW 30 000 for the cucumber enzyme and 35 000 for that of the squashes. The other two forms in the cucumber appear to be a dimer and a tetramer, whilst a tetramer, an octamer, a dodecamer, and a polymer of MW between 670 000 and 2 000 000 are likely to be the other four forms present in the squashes. The monomer was the most abundant form in the cucumber and the tetramer in the two squashes. The peel of these fruits was higher in activity than the flesh, but the isozyme pattern was the same in peel and flesh. The tetramer of the squashes and the dimer of cucumbers were the most resistant forms to heat inactivation. The enzyme is soluble and not associated with subcellular particles.     

Ascorbate oxidase is the potential conductor of a symphony of signaling pathways

The functional role of ascorbate oxidase (AO; EC 1.10.3.3) has never been fully explained so far, due to the difficulties in understanding the presence of an enzyme specifically oxidizing ascorbate with no obvious advantage, and the apparent disadvantage of lowering plant stress resistance as a consequence of ascorbate consumption. Here we suggest a complete change of perspective, by proposing an essential role of AO as a modulator of both ascorbate and oxygen content, with relevant implications related to signaling. By affecting the overall redox state, AO is actually involved in redox regulation in the extracellular matrix. In addition, AO can contribute to creating a hypoxic microenvironment, especially relevant in the maintenance of meristem identity and the establishment of mutualistic plant-microbe interactions. We also hypothesize the possible involvement of AO in the activation of a signaling cascade analogous to the mechanism of prolyl hydroxylases/Hypoxia Inducible Factors in animals.     

Metabolic networks of Cucurbita maxima phloem

Metabolomic analysis aims at a comprehensive characterization of biological samples. Yet, biologically meaningful interpretations are often limited by the poor spatial and temporal resolution of the acquired data sets. One way to remedy this is to limit the complexity of the cell types being studied. Cucurbita maxima Duch. vascular exudates provide an excellent material for metabolomics in this regard. Using automated mass spectral deconvolution, over 400 components have been detected in these exudates, but only 90 of them were tentatively identified. Many amino compounds were found in vascular exudates from leaf petioles at concentrations several orders of magnitude higher than in tissue disks from the same leaves, whereas hexoses and sucrose were found in far lower amounts. In order to find the expected impact of assimilation rates on sugar levels, total phloem composition of eight leaves from four plants was followed over 4.5 days. Surprisingly, no diurnal rhythm was found for any of the phloem metabolites that was statistically valid for all eight leaves. Instead, each leaf had its own distinct vascular exudate profile similar to leaves from the same plant, but clearly different from leaves harvested from plants at the same developmental stage. Thirty to forty per cent of all metabolite levels of individual leaves were different from the average of all metabolite profiles. Using metabolic co-regulation analysis, similarities and differences between the exudate profiles were more accurately characterized through network computation, specifically with respect to nitrogen metabolism.     

Purification and properties of glycollate oxidase from Pisum sativum leaves

A rapid and efficient method for the isolation of glycollate oxidase from pea leaves is described. The method utilizes the unusually high isoelectric point (pH 9·6) which has been determined for the enzyme using isoelectric focusing. The enzyme is apparently homogeneous by polyacrylamide gel electrophoresis and has a MW of ca 100000. Some properties of the enzyme are described.     

Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties.

1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.     

Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.     

Kinetic properties of IAA oxidase from mung bean cotyledons

A crude enzyme preparation from mung bean cotyledons was separated into peroxidative and non-peroxidative IAA oxidase on a DEAE-cellulose column. Both fractions differed in their pH optima, K_m and V_max. The K_m and V_max of non-peroxidative IAA oxidase were higher than those of peroxidative IAA oxidase. Peroxidative IAA oxidase showed a linear increase in absorption at 247 and 254 nm after a short lag of 2–3 min. The addition of catalytic amounts of hydrogen peroxide eliminated the lag period and also enhanced the rate of IAA degradation. The non-peroxidative IAA oxidase fraction, however, did not exhibit any significant increase in absorption at 247 and 254 nm and showed a lag period of 5 min which was not affected by hydrogen peroxide. Instead, addition of the same catalytic amount of hydrogen peroxide inhibited the rate of IAA degradation. The peroxidative IAA oxidase fraction exhibited the reaction kinetics characteristic of peroxidase-catalysed IAA degradation. The rate of IAA oxidation by purified non-peroxidative IAA oxidase was very low. The slow rate of catalysis shown by non-peroxidative IAA oxidase appears to be due to the presence of inhibitor(s).     

Ascorbate peroxidase activity of cytochrome c

Abstract

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10–60 µM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions.     

A 24β-ethyl-Δ7-steryl glucopyranoside from Cucurbita pepo seeds

In the seeds of Cucurbita pepo three closely related 24-ethyl-Δ⁷-steryl glucosides were identified by hydrolytic studies and spectral analysis as spinasteryl-β-D-glucopyranoside, the new 3-O-(β-D-glucopyranosyl)-24β-ethyl-5α-cholesta-7,25(27)-dien-3β-ol and the corresponding Δ^22E,25,(27)-trienol. Except for its occurrence in cucumber seeds the latter is so far unknown as a natural product.     

Structure and biochemistry of phloem-proteins isolated from Cucurbita maxima

Phloem proteins (P-proteins) were isolated from exudates of both the fruit skin and the stem of the pumpkin, Cucurbita maxima, and the cucumber, Cucumis sativus, and were purified by ammonium sulphate precipitation and chromatography over Sephadex, DEAE-cellulose, and CM-cellulose. It was found that the fruit skin phloem is an excellent source for large-scale preparations of P-proteins which are biochemically and structurally identical with those from stem exudates. Besides fractionation on columns the P-protein preparation was characterized by electrophoresis in polyacrylamide gels (in SDS or acidic urea solutions) and on cellulose acetate, by precipitation studies using salts, acids and thiol reagents, by centrifugation techniques, by determination of amino acid composition, by cyanogen bromide cleavage, by immunodiffusion and immunoelectrophoresis using anti-P-protein-sera obtained from mice, and by electron microscopy of negatively stained and ultrathin sectioned preparations of native and reaggregated P-proteins. P-proteins were identified as a class of at least eight different but related basic (IEPs from pH 9.6 to 10.4) proteins and polypeptides of molecular weights 14000, 17000, 44000, 59000, 116000 and 158000 D which are rich in lysine, leucine, glycine, glutamic acid (plus glutamine) and aspartic acid (plus asparagine). Structurally they were pleomorphic and formed, at various proportions, floccules, fibrils, doughnuts and 100 Å lamellae with a membrane-like ultrastructure. The P-proteins showed in several properties (amino acids, IEPS, retention on CM-cellulose, antigenic sites, molecular weights of the smaller components) a strong similarity to proteins extracted from a ribosomal fraction prepared from cucurbit seedlings, in particular to a weakly basic subtraction of ribosomal proteins. It is hypothesized that P-proteins are formed during sieve element maturation by aggregation and oxidative disulphide cross-linking of preexisting proteins which, at least in the Cucurbitaceae, are basic and may include ribosomal or ribosome-associated proteins. Possible functions of these aggregates are discussed.     

Multiple forms of Vitis vinifera catechol oxidase

Grape catechol oxidase shows multiple forms upon ion exchange chromatography, acrylamide gel electrophoresis and gel filtration. Conversion of some bands into others, which occurs during isolation and storage, is enhanced by dilution and by treatment with urea or acid pH. Estimation of MWs suggested that the conversions might be due to dissociation of the enzyme into subunits, but attempts to induce reassociation were unsuccessful. The effects of urea and acid pH could be imitated by partial enzymic proteolysis of the enzyme. Analysis of the various bands observed in gel electrophoresis suggested that some of the enzyme forms have the same MWs but differ in charge distribution.     

Different AT-rich satellite DNAs in Cucurbita pepo and Cucurbita maxima

Summary The AT-rich highly repeated satellite DNA of Cucurbita pepo (zucchini) and Cucurbita maxima (pumpkin) were cloned and their DNA structure was investigated. DNA sequencing revealed that the repeat length of satellite DNA in Cucurbita pepo is 349–352 base pairs. The percentage of AT-base pairs is about 61%. This satellite is highly conserved in restriction enzyme pattern and DNA sequence; sequence heterogeneity is about 10%. In contrast, the satellite DNA of Cucurbita maxima has a repeat length of 168–169 base pairs. This satellite is also rich in AT-base pairs (64%), existing in at least three different variants as revealed by restriction enzyme analysis and DNA sequencing. The sequence heterogeneity between these variants is about 15%. The two satellite DNAs showed no cross-hybridization to each other and sequence homology is only limited. Nevertheless, we found in the C. pepo genome a high amount of sequences resembling the satellite of C. maxima. In contrast, the satellite repeat of C. pepo is found in the C. maxima DNA only in a few copies. These observations were discussed with respect to satellite DNA evolution and compared to the data received from monocotyledonous species.     

Cloning and expression of peroxisomal Ascorbate Peroxidase gene from wheat

A full-length cDNA encoding wheat peroxisomal ascorbate peroxidase (pAPX) was cloned by Suppression Subtractive Hybridization (SSH) and in silico approach. The cDNA was 1027 bp in length and contained a complete ORF of 876 bp, which encodes a protein of 292 amino acid residues. Its deduced amino acids sequence had 84% identity with that of pAPX from barley. The gene was designated as Ta-pAPX. The Ta-pAPX homologous genes were mapped on wheat chromosome 7A and 7D using Chinese Spring nulli-tetrasomic lines analysis. Northern analysis indicated that, after inoculation by Erysiphe graminis Dc.f.sp. tritici, the expression of Ta-pAPX gene in Yangmai5 was enhanced, but its expression in wheat-Haynaldia villosa 6VS/6AL translocation lines changed a little. The results implied that Ta-pAPX may be related to susceptibility of wheat to powdery mildew. The complete coding sequence of Ta-pAPX was cloned into an expression vector pET32 (a+) and a protein with the same deduced molecular weight (MW) was expressed in E. coli BL21 (DE3), which showed ascorbate peroxidase activity.     

Isolation and characterization of catechol oxidase from Solanum melongena

Catechol oxidase was distributed in soluble and particulate fractions of Solanum melongena. The purified preparation appears to be homogeneous by polyacrylamide gel electrophoresis. The enzyme shows two pH maxima—with catechol, 6.5 and 7.5; and with dopa, 6.5 and 7.9. The latent form of the enzyme does not occur in S. melongena. The preparation resembles the enzyme from other sources in substrate specificity towards various mono- and diphenols, having a higher affinity for catechol than dopa; this tendency increases on purification. The cresolase activity decreases with purification and a lag period with p-cresol is observed. The oxidation of mono- and diphenols is inhibited by ascorbic acid, sulphydryl compounds and chelating agents.     

Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima extract

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 degrees C using initial ascorbic acid concentration in the range 50-750 M allowed estimating the Michaelis constant for this substrate (Km = 126 microM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (DeltaH* = 10.3 kJ mol-1; DeltaG* = 87.2 kJ mol-1; DeltaS* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long-term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10-70 degrees C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (DeltaH*D = 51.7 kJ mol-1; DeltaG*D = 103 kJ mol-1; S*D = -160 J mol-1 K-1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half-life (1047 min at 10 degrees C and 21.2 min at 70 degrees C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid-liquid extraction techniques.     

Filament formation in vitro of a sieve tube protein from Cucurbita maxima and Cucurbita pepo

Summary Phloem proteins of the sieve tube exudate from Cucurbita maxima Duch. and Cucurbita pepo L. were investigated as to their filament forming ability in vitro. From the two main proteins (116000 dalton, 30000 dalton) only the 116000 dalton protein was found to form reversibly distinct filaments of 6–7 nm diameter upon removal of SH-protecting agents from the buffer, whereas the 30000 dalton protein was precipitated as amorphous material under these conditions. The protein filaments were similar to the filaments ocurring within the sieve tube cells in vivo.Abbreviations SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

环境胁迫和抗坏血酸的氧化还原状态

为了解环境胁迫对植物体中抗坏血酸含量及氧化还原状态的影响，以不同强度的冰冻和干旱两种胁迫为例，研究了它们对沈阳几种针叶树离体叶抗坏血酸、脱氢抗坏血酸含量以及抗坏血酸-谷胱甘肽循环中4种酶活性的影响。结果表明，两种胁迫达到一定强度后，都能使还原态抗坏血酸含量下降而使脱氢抗坏血酸含量上升。冰冻使抗坏血酸过氧化酶和单脱氢抗坏血酸还原酶活性下降。轻度失水使这两种酶活性上升，失水加重后转而趋于下降。脱氢抗坏血酸还原酶和谷胱甘肽还原酶活性对两种胁迫反应均不如前两种酶敏感。结合以前的研究结果，认为这一H2O2清除系统在导致驯化(acclimation)的轻度胁迫作用下可以得到加强，而当胁迫强度过大时则其清除能力下降并使组织受到伤害。文中还报告了沈阳几种针叶树抗寒性和针叶中抗坏血酸含量及上述4种酶活性之间的相关关系。