María Estrella Legaz

Effect of polyamines on the separation of ovalbumin glycoforms by capillary electrophoresis.

The successful separation of ovalbumin (M(r) 45,000; pI 4.7) glycoforms by capillary electrophoresis in an uncoated fused-silica capillary with different buffer additives is reported. The optimum conditions for obtaining the resolution of glycoforms were 25 mM borate (pH 9.0) containing 0.87 mM spermidine or 0.14 mM spermine. The effects of different concentrations of putrescine, cadaverine, spermidine, spermine and some monoamines or diamines are compared in terms of selectivity factors of ovalbumin peaks. Addition of sodium dodecyl sulfate at a concentration below the critical micelle concentration increased the resolution between the three main peaks of ovalbumin but did not permit their microheterogeneity to be expressed.

A role for sugarcane glycoproteins in the resistance of sugarcane to Ustilago scitaminea

Smut is a major disease of sugarcane caused by Ustilago scitaminea. Germination of fungal teliospores is achieved on the internode surface of plants, and it is followed by the formation of appressoria. A primary response of sugarcane plants to the infection seems to be the production of several glycoproteins, defined as mid-molecular mass (MMMG) or high molecular mass (HMMG) macromolecules. Teliospore germination in the presence of both MMMG and HMMG decreased about 50% following 5 h of teliospore contact with glycoproteins. This may be related to the ability of glycoproteins to produce cytoagglutination. Binding of fluorescein-labelled glycoproteins was studied by fluorescence microscopy, showing that staining of cells was not uniform, but mainly in the contact zone between two individual teliospores when aggregated. HMMG was composed of only one fraction that was completely retained by smut teliospores, whereas three of the five different glycoproteins occurring in the MMMG fraction were retained by teliospore cell walls. Moreover, a unique application of salicylic acid, naturally produced by sugarcane stalks after experimental fungal infection, enhanced the production of both glycoprotein pools. A hypothesis about the role of both HMMG and MMMG as defence glycoproteins is discussed.

Role of polyamines in the infection of sugarcane buds by Ustilago scitaminea spores

Experimental infection of sugarcane buds from sensitive (Barbados 42231) and resistant (Mayari 5514) cultivars of sugarcane hybrids to smut with teliospores of Ustilago scitaminea leads to a remarkable increase of both free and conjugated polyamines. Whereas resistant buds mainly produce free putrescine, the sensitive cultivars produce acid soluble-conjugated as well as acid insoluble-conjugated spermidine and spermine after infection. A net maximum of both polyamines is reached 13 or 17 d after infection and levels decrease later. This maximum after infection is always higher than that found for non-infected buds. Variation of cadaverine, also produced by sugarcane buds, does not show any clear correlation with smut development.

Pigment analysis of sun and shade populations of Cladonia verticillaris

Abstract The natural occurrence of the lichen Cladonia verticillaris as sunny and shaded populations implies changes in the pigment content. Shade specimens contain more chlorophyll a and b as well as more α- and β-carotene than those found in unshaded lichens. However, lichens growing in sunny locations contain more methyl β-orcinol carboxylate, orcinol and fumarprotocetraric acid than those found in lichens growing under low light intensities. No structural differences in the chloroplasts of the two species bave been detected.

Ionic adsorption of catalase on bioskin: kinetic and ultrastructural studies.

Bioskin is a natural polymer produced by Acetobacter xylinum and several yeasts in culture. It contains glucosamine and N-acetyl galactosamine which promote ionic adsorption of catalase at the adequate pH value. High values of ionic strength are required to enzyme desorption. Adsorption of catalase on bioskin fibers has been visualized by scanning electron microscopy associated to a dispersion X-ray analyzer. At low enzyme density, the affinity of the immobilized catalase for hydrogen peroxide was 30% lower than that of the free enzyme. This affinity decreased dramatically at higher density of immobilized enzyme and could not be increased by agitation of the enzyme reaction mixture. Immobilized catalase retains about 70% of its initial activity after 16 d storage, whereas soluble enzyme is completely inactivated after 3 d at room temperature. The haeme group of catalase is not protected after immobilization since it is accessible to both EDTA and phloroglucinol, chelating agents which inactivate catalase by removing the iron atom from the haeme group.

Separation of soluble glycoproteins from sugarcane juice by capillary electrophoresis

Abstract Two different pools of glycoproteins, tentatively described as high and mid-molecular mass polymers, are currently separated by conventional gel filtration. Anion-exchange chromatography on DEAE-Sephadex A-60 discriminates between neutral or cationic, and anionic polymers composing both fractions. The occurrence of both cationic and anionic glycoproteins has been corroborated after separation by capillary electrophoresis. Total or partial digestion of the glycosidic moiety of these glycoproteins using β-1,2-fructofuranosidase reveals that cationic species from the mid molecular mass glycoproteins move to the neutral or anionic zone of the electropherogram whereas only one cationic form from high molecular mass polymers remains unchanged after enzymatic hydrolysis of the glycosidic moiety. This indicates that this moiety is able to modify the net charge of some of the mid-molecular mass glycoproteins whereas the net charge of cationic high molecular mass polymer can be due to the own protein moiety or, probably, to some polycationic polyamines bound on this moiety.

Separation of acidic proteins by capillary zone electrophoresis and size-exclusion high-performance liquid chromatography : a comparison

Abstract Successful separations of alcohol dehydrogenase (pI = 5.4), β-amylase (pI = 5.2) and albumin (pI = 4.7) by capillary electrophoresis in uncoated fused-silica capillaries are reported. Different electrophoretic conditions, consisting in variation of temperature, applied voltage and ionic strength of the buffer used as electrolyte, were tested in order to compare the separation efficiency, resolution and selectivity of the acidic proteins. The results were compared with those obtained by size-exclusion chromatography. Rinsing of the capillary between runs, in order to eliminate adsorbed proteins, can shorten its useful lifetime.

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.

Soluble glycoproteins from sugar cane juice analysed by high-performance liquid chromatography and fluorescence emission

Abstract Soluble polysaccharides have been isolated from sugar cane by size-exclusion chromatography using two successive columns of Sephadex G-10 and G-50. Two main fractions of different molecular mass were separated and their homogeneity was tested by HPLC using a Zorbax GF-450 and a Zorbax GF-250 column in series. Whereas the high-molecular-mass soluble polysaccharide (SP) fraction seems to be composed of two different polymers with retention times of about 16.5 and 24.5 min, the mid-molecular-mass carbohydrate (MMMC) fraction is resolved as only one peak with a retention time of about 2.45 min. This implies that Sephadex G-50 is not able to discriminate between the two categories of macromolecules, as SP eluted from Sephadex G-50 is clearly contaminated by MMMC. Fractionation of clarified juice with 80% (v/v) ethanol separates SP as an insoluble fraction and MMMC as an ethanol-soluble macromolecule. The natural fluorescence of both polymers reveals that they are glycoproteins and, in addition, they can clearly be recognized by the degree of tryptophan exposure to its chemical microenvironment.

A Simple Assay Demonstrating the Effect of Rehydration on the Orsellinate Depside Hydrolase Activity of Evernia prunastri

A new, simple assay of orsellinate depside hydrolase (EC. 3.1.1.40) by high performance liquid chromatography is described. Enzymatic hydrolysis of evernic acid produces equimolar amounts of both orsellinic and everninic acids. Evernia prunastri thallus has a pre-existent, partially inactive hydrolase, which is activated after rehydration of the thallus.