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The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis.

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.

Amino Acid Sequence↗

Serine proteinase from Cucurbita ficifolia seeds.

A new serine proteinase was isolated from Cucurbita ficifolia seeds by the purification procedure, which includes: extraction, salting out with ammonium sulphate, chromatography on CM-cellulose. Sephacryl S-300 gel filtration and h.p.l.c. on DEAE-2SW TSK column. The enzyme was homogeneous both in native and SDS PAGE. Three independent methods showed its molecular mass to be approximately 77 kDa. The enzyme was inhibited by specific serine proteinase organic inhibitors, and was active in the presence of inhibitors specific for other proteinase classes. Surprisingly, squash proteinase exhibited a very high and broad pH optimum with a maximum at 10.7. It hydrolysed many different peptide bonds in B-chain of insulin and was able to cleave four bonds in endogenous serine proteinase inhibitor (CMTI).

Amino Acid Sequence↗

[Characterization of Xanthomonas campestris pv. cucurbitae, causal agent of the bacterial spot of squash].

Bacterial leaf spot of squash was characterized for the first time in Argentina. Cultural, physiological, morphological and cross-infection tests on Cucurbitaceae showed that the pathogen was Xanthomonas campestris pv. cucurbitae (Bryan) Dye. The bacterium isolated from winter squash proved pathogenic for pumpkin, winter squash, cucumber and watermelon but no for muskmelon.

Plant Diseases↗

Peptide hydrolase activities in seedlings and hormone-treated cotyledons of pumpkin (Cucurbita pepo).

Enzymes hydrolyzing Gly-Ala-, Met-Met- and Pro-4-phenylazo-phenylamides, and N-benzoyl-L-arginine-4-nitroanilide have been identified in germinating seeds and cotyledons of pumpkin (Cucurbita pepo). The enzyme activities per cotyledon increase markedly during the germination process, but the proportion of enhancement depends on the type of enzyme species. The increase in enzyme activities is due to de novo synthesis as shown by cycloheximide treatment and is influenced by phytohormones (cytokinins and abscissic acid). In isolated cotyledons exogenous cytokinin (benzyladenine) obviously can replace the effect of the embryo as the source of endogenous hormone. Abscissic acid counteracts the cytokinin effect. It is suggested that aminopeptidases have a biological function in reserve protein degradation of the cotyledons during seed germination. Our results do not support the assumption that the embryonic axis of the growing seedling serves as a "sink" of proteolytic products resulting in an activation of peptide hydrolases in the cotyledons, but rather de novo synthesis of these enzymes seems to be controlled by substances (phytohormones) originating from the embryo.

Aminopeptidases↗

Trypsin inhibitors in summer squash (Cucurbita pepo) seeds. Isolation, purification and partial characterization of three inhibitors.

Three trypsin inhibitors were isolated from summer squash (Cucurbita pepo) seeds and purified to homogeneity by fractionation with ammonium sulphate and methanol, ion-exchange chromatography and gel filtration. All three inhibitors have lysine at their active site. Two of them (II, IV) show the same isoelectric point (at pH 5.6), amino acid composition and molecular mass (3259). The third inhibitor (III) of molecular mass of 3654 and isoelectric point at 4.9 has additionally one histidine residue and two glutamic acid residues more per molecule.

Amino Acids↗

Isolation and partial amino acid sequence of the trypsin inhibitor from the seeds of Cucurbita maxima.

Trypsin inhibitor from squash (Cucurbita maxima) seed was extracted with 0.1 M-acetate buffer, pH 4.5, purified on immobilized trypsin, and separated by SE-Sephadex C-50 chromatography into three active fractions. All of them inhibited trypsin to the same extent, showed no antichymotrypsin or antikallikrein activity, had a similar molecular weight (about 3300), and contained no tryptophan, phenylalanine or threonine. The partial amino acid sequence of tryptic and peptic peptides of fraction III was determined by Edman degradation procedure.

Amino Acid Sequence↗

[Effects of Celosia argentea and Cucurbita moschata extracts on anti-DNP IgE antibody production in mice].

We have already reported that the Perilla frutescens extract (PFE) suppressed anti-DNA IgE antibody production in mice. In this study, we prepared extracts of Celosia argentea L. (CAE) and Cucurbita moschata Duch (CME), which are Chinese herbal medicines like Perilla frutescens, and examined the effects on anti-DNP antibody responses in mice. To examine the effects of CAE & CME on primary antibody responses, CAE & CME were intraperitoneally injected the day before primary immunization of DNP-ovalbumin. Anti-DNP antibody production was markedly suppressed. Then, we examined the effects on secondary antibody responses. CEA & CME were injected only the day before secondary immunization. Anti-DNP IgE production was markedly suppressed, but IgG responses were not affected. It was also found that mitogenic activity occurred in CAE & CME dose dependently in vitro. These effects of CAE & CME were superior to that of PFE. These results suggest that CAE & CME may be more useful than PFE for the suppression of IgE antibody in certain allergic disorders.

Animals↗

Identification of the proteins responsible for SAR DNA binding in nuclear matrix of Cucurbita pepo.

The nuclear matrices from White bush (Cucurbita pepo var. patisonina) cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human beta-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments.

Antigens, Nuclear↗

Changes in DNA methylation during somatic embryogenesis in Cucurbita pepo L.

Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 m M NH(4)Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 micro M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.

2,4-Dichlorophenoxyacetic Acid↗

Single step determination of fragrances in Cucurbita flowers by coupling headspace solid-phase microextraction low-pressure gas chromatography-tandem mass spectrometry.

Coupling headspace solid-phase microextraction (HS-SPME) and low-pressure gas chromatography-tandem mass spectrometry (LP-GC-MS-MS) has been used for determining 20 volatile compounds present in flowers. HS-SPME coupled with LP-GC-MS-MS acts in a synergic way allowing a fast extraction and analysis of the target compounds. The method has been optimised studying the influence of the adsorption temperature and adsorption time. The best results were obtained heating the SPME vials at 60 degrees C for 5 min using 65 microm poly(dimethylsiloxane-divinylbenzene) fibers. The validation of the method ensures the fitness for the purpose of the analytical method, achieving appropriate lower limits, recoveries and precision. The analytical method has been applied to the characterisation of zucchini flowers fragrances in air using passive sampling, in order to improve our knowledge on zucchini fragrances and to better pollination technique in future steps.

Cucurbita↗

Effect of infiltrated polyamines on polygalacturonase activity and chilling injury responses in zucchini squash (Cucurbita pepo L.).

The effect of exogenous polyamines on electrolyte leakage, chilling index, polygalacturonase activity (PG), ethylene production, and firmness in zucchini squash fruits stored for 12 days at 2 degrees C or 10 degrees C, 85-90% RH was evaluated. Fruits were infiltrated with putrescine (PUT) spermidine (SPD) and spermine (SPM) at 0.1, 0.25, 0.5, 2.0, and 4.0 mM. All polyamines exerted a protective effect on cell and organelle membranes. The most effective was SPD, which reduced electrolyte leakage between 62% and 82%, compared to control fruits stored at 2 degrees C. At 10 degrees C they did not exhibit chilling injury (CI) symptoms, while at 2 degrees C SPM (0.5 mM) and SPD (0.5 mM) diminished them 92% and 100%, respectively; which extended storage life for 8-10 days at 2 degrees C. High concentrations of polyamines (>2.0 mM) caused the appearance of CI symptoms. PG activity diminished proportionally to the concentration of polyamine except for the concentration at 4.0 mM. No significant changes were observed in ethylene production.

Cold Temperature↗

Irreversibility of chilling injury in zucchini squash (Cucurbita pepo L) could be a programmed event long before the visible symptoms are evident.

Zucchini fruits were subjected to 2.5 or 10 degrees C for 16d, followed by transfer to 20 degrees C for 24h in order to evaluate the relationship between ripening pattern, measured as CO(2) evolution and ethylene (C(2)H(4)) production, and metabolic heat production (q). Chilling injury (CI) visible symptoms were evident after 8d at 2.5 degrees C, but none were recorded on fruits kept at 10 degrees C. In fruits held at 10 degrees C, q, C(2)H(4) production, and CO(2) evolution diminished in the course of 16d, whereas in those at 2.5 degrees C CO(2) evolution showed an early burst peaking at 8d. Both C(2)H(4) production and q also showed a burst at 2.5 degrees C but they started at 4 and 8d, respectively, and peaked at 12d. The results showed that irreversibility of chilling injury in zucchini could occur long before the appearance of visible symptoms, although the metabolic activity accompanying the irreversibility process was not noticeable by isothermal calorimetry.

Calorimetry, Differential Scanning↗

Role of organic acids in enhancing the desorption and uptake of weathered p,p'-DDE by Cucurbita pepo.

Experiments were conducted to assess the effect of seven organic acids [succinic, tartaric, malic, malonic, oxalic, citric, ethylene-diaminetetraacetic (EDTA)] over a concentration range of two orders of magnitude (0.001-0.10 M) on the abiotic desorption of weathered p,p'-DDE and the extraction of polyvalent inorganic ions from soil. At 0.05 M all organic acids significantly increased contaminant desorption by 19-80%. Organic acids also increased the aqueous concentration of eight inorganic constituents extracted from soil, with at least a six-fold increase in the release of Al, Fe, Mn, and P at 0.001 M. Zucchini seedlings grown for 28 d in soil containing weathered p,p'-DDE (300 ng/g, dry weight) were periodically amended with distilled water, citric or oxalic acids (0.01 M). Plants receiving water removed 1.7% of the p,p'-DDE from the soil. Seedlings amended with citric or oxalic acids removed 2.1 and 1.9% of the contaminant, respectively, and contained up to 66% more contaminant in the shoot system than unamended vegetation. A second crop of untreated (distilled water) zucchini in the same soil removed more contaminant than the first crop (2.5%), although the addition of organic acids did not further enhance contaminant uptake. The data indicate that the addition of low molecular weight organic acids causes the partial dissolution of the soil structure through the chelation of inorganic structural ions, potentially enhancing bioavailability and having implications for the phytoremediation of persistent organic pollutants in soil.

Acids↗

Purification and characterization of moschins, arginine-glutamate-rich proteins with translation-inhibiting activity from brown pumpkin (Cucurbita moschata) seeds.

From fresh brown pumpkin seeds, two proteins with a molecular mass of 12kDa and an N-terminal sequence rich in arginine and glutamate residues were obtained. The protein designated alpha-moschin closely resembled the fruitfly programmed-cell death gene product and the protein designated beta-moschin demonstrated striking similarity to prepro 2S albumin in N-terminal sequence. alpha- and beta-moschins inhibited translation in the rabbit reticulocyte lysate system with an IC(50) of 17 microM and 300nM, respectively.

Amino Acid Sequence↗

Influence of cultivar, cooking, and storage on cell-wall polysaccharide composition of winter squash (Cucurbita maxima).

Changes in the cell-wall polysaccharides (CWP) of the edible tissues of four winter squash cultivars during storage and after cooking were investigated. A procedure for isolating cell walls of tissues containing high levels of starch was used. The starch-free CWP were sequentially fractionated using CDTA, dilute Na(2)CO(3), and 4 M KOH. Cellulose made up 40-42% of the total CWP for three cultivars (Delica, CF 2, and CF 4) at harvest but was 35% in the softer Red Warren. The pectic polysaccharides of Delica, CF 2, and CF 4 cell walls are more branched than those from Red Warren squash. The higher proportion of uronic acid in the pectic polysaccharides of Red Warren squash correlates with its lower firmness. Cooking resulted in an increase in the water-soluble pectins and a decrease in the pectins associated with cellulose. The total CWP content of the squash cultivars remained unchanged for up to 2 months of storage and then markedly decreased between 2 and 3 months of storage. The galactose content of Delica and Red Warren cell walls remained relatively constant from harvest to 2 months of storage and then decreased markedly during 2-3 months of storage.

Carbohydrates↗