Nadežda Kolarova

Light-stimulated phosphorylation of proteins in cell-free extracts from Trichoderma viride

When illuminated by visible light, cell‐free extracts from the fungus Trichoderma viride catalysed the phosphorylation of at least two proteins with molecular masses of 18 and 114 kDa which were practically absent when the phosphorylation was performed in the dark. The effect of light could be substituted by 3mM cyclic AMP, not only in the cell‐free extract, but also in the separated cytosol. It is concluded that the process of photoinduced conidiation in Trichoderma involves phosphorylation of conidiation‐specific proteins by (a) cyclic AMP‐dependent protein kinase(s) present in the cytosol.

Conjugation of yeast mannans with protein employing cyanopyridinium agent (CDAP) - an effective route of antifungal vaccine preparation

The possibility of using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for activation of saccharide hydroxyl groups (instead of hazardous cyanogen bromide) is examined with cell-surface mannans of the yeasts Candida albicansCandida tropicalis,Candida lambicaand galactoglucoxylomannan of Cryptococcus laurentii.Direct conjugation with human serum albumin yielded soluble products with increased molecular size in comparison with the original polysaccharides. Immunodiffusion experiments revealed that conjugation did not affect the immunospecificity of the antigen epitope.

Hyperpolarization and intracellular acidification in Trichoderma viride as a response to illumination.

Using indirect methods based on uptake of [3H]tetraphenylphosphonium cation and [14C]benzoic acid by cells of the fungus Trichoderma viride we found that the illumination-induced transient hyperpolarization of the plasma membrane is followed immediately by a rapid temporary decrease in intracellular pH. Hyperpolarization and intracellular acidification were completely suppressed by 150 mM-KCl and by the K(+)-ionophore valinomycin. The light-induced acidification of the cytoplasm was not observed in the presence of the cytochrome respiratory chain inhibitors antimycin A and mucidin. Based on these results, we hypothesize that the hyperpolarization of the cells is the consequence of an efflux of K+ through a light-activated K(+)-channel in the plasma membrane. The loss of positive charge in the cytoplasm caused by this efflux of cations is counterbalanced by H+ originating from the light-activated mitochondrial respiratory chain.

The glycoprotein character of multiple forms of Aspergillus polygalacturonase.

Comparisons of known primary structures of polygalacturonases show that extent and localization of potential N-glycosylation sites differ. Some sites are similar in position and adjacent to strictly conserved residues at the potential active site. The presence of N-acetylglucosamine and mannose in the molecules of two homogeneous, major Aspergillus sp. polygalacturonase forms was confirmed by IR spectroscopy. The purification method, based on interaction of the carbohydrate part with concanavalin A immobilized on chlorotriazine bead cellulose, was optimized. Deglycosylation with N-glycosidase F under denaturating and nondenaturating conditions led to molecular mass decreases followed by complete inactivation of the polygalacturonase enzyme activity. These results show the importance of glycosylation in these protein forms, while the comparative patterns establish both variability and some similarities in overall glycosylation architectures.

Characterization of cellulolytic enzyme complexes obtained from mutants of Trichoderma reesei with enhanced cellulase production

SummaryThe cellulolytic enzyme complexes secreted by the fungus Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15, and MHC 22 were characterized by determining their specific filter-paper (FP)-, carboxymethylcellulase (Cx)-and β-glucosidase (βG)-activities. They were characterised further by measuring their Cx and βG profiles after separation on an isoelectrofocusing column over the pH range 3–10. While the overall FP-activity was roughly equal in all preparations, the specific β-glucosidase activity was highest in mutants MHC 15 and MHC 22 which are distingiushed morphologically from the parent strain, QM 9414, by a higher degree of branching of their hyphae. Two peaks of β-glucosidase activity were detected by isoelectric focusing in preparations from QM 9414 and M 6, none in the enzyme from the mutant M 5 while 3 and 4 peaks respectively were found in preparations from morphological mutants MHC 15 and MHC 22. The higher β-glucosidase activity in these last two preparations was also reflected in the higher glucose to cellobiose ratio in the initial stages of cellulose hydrolysis by the individual enzyme preparations.

Structure of Glucomannan-Protein from the Yeast Cryptococcus Laurentii

Abstract The structure of an extracellular glucomannan-protein produced by Cryptococcus laurentii was studied. The glucomannan-protein was isolated via its insoluble copper complex. It was homogeneous on free-boundary electrophoresis, contained 91% saccharide, 6.5% protein and 1% phosphorus. It had Mn 21,000. The carbohydrate portion was composed of D-mannose and D-glucose in 33:2 molar ratio. From the results of compositional and methylation analyses, conventional acetolysis, as well as 1H and 13C NMR spectroscopy it was concluded that the glucomannan has an α-(1→6)-linked D-mannopyranosyl backbone having most residues (about 83%) substituted at O-2 with one, two, three or four D-mannopyranosyl units connected by α-(1→2) and α-(1→3) linkages. Moreover, an additional side chain with the α-D-Manp-(1→3)-D-Manp-(1→2)-D-Manp-(1→2)-D-Manp-D-Manp backbone structure in which α-D-glucopyranose residue is linked to O-2 of the mannopyranose unit next to the reducing end. Alkali treatment of glucomannanprotein in th...

Affinity Chromatography of Glycoenzymes and Glycoproteins on Concanavalin A-Bead Cellulose

Abstract Concanavalin A immobilized on chlorotriazine bead cellulose was applied to affinity purification of glycoenzymes and glycoproteins. Enzymes such as invertase from bakers yeast, endopolygalacturonase (Rohament P) and exopolygalacturonase from carrot roots, as well as extracellular mannoproteins from the yeast Cryptococcus laurentii were examined. Chromatography was performed on minicolumns filled with Concanavalin A-triazine bead cellulose gel with the content of immobilized Concanavalin A within the range 1.2 – 8.2 mg per mL of gel. The specifically bound glycoenzymes or glycoproteins were eluted with a solution of the corresponding counter-ligand a-methyl mannopyranoside. Individual degrees of purification, estimated from the measurements of specific activity of crude and purified glycoenzymes, were 14.5-fold for invertase, 93-fold for polygalacturonase and 3.9-fold for exopolygalacturonase. The yeast mannoprotein was isolated from the heteroglycoprotein fraction. The purified mannoprotein cont...

AN EXTRACELLULAR GALACTOGLUCOXYLOMANNAN PROTEIN FROM THE YEAST Cryptococcus laurentii VAR. laurentii

An extracellular galactoglucoxylomannan protein composed of d-galactose, d-glucose, d-xylose and d-mannose in a 2.9:1.0:1.1:10.2 mole proportion has been isolated from culture medium of Cryptococcus laurentii var. laurentii. The polymer of number average molecular mass 19,000 contained 86% carbohydrates, 6.5% protein and 0.7% phosphorus. Results of structural analyses suggested a highly branched comb-like structure of the polysaccharide with a backbone composed of 6-linked α-d-Man residues. Mannose units of the backbone are highly branched at O-2, O-3, and O-4 by side chains composed mainly of 2-linked α-d-Man mostly in the form of dimers and trimers, and to a lesser amount as tetra- and pentamers. Galactosyl units were found to be mostly 6-linked with a very low degree of substitution. Mannose side chains are further branched with d-Xyl, d-Glc, and d-Gal residues preferably in β their forms. The protein part of the glycoprotein was O-glycosylated by mannose, mannobiose, and mannotetrose.

Sensitivity of various yeasts to crude cellulolytic enzyme complexes fromTrichoderma reesei

SummaryTwenty-six yeast strains, representative of different yeast genera, were tested for their sensitivity to crude extracellular cellulolytic enzyme complexes obtained from the fungusTrichoderma reesei QM 9414 and its mutants M 6 and MHC 22 (Microcrystalline cellulose was the sole carbon source.) Practically all the yeast strains tested were found to be sensitive, exhibiting signs of cellwall weakening and lysis during prolonged incubation with the emzymes fromTrichoderma. Under growth conditions, the effect of cellulolytic enzymes on yeast cells and their growth rates was much less pronounced. However, at increased cellulase concentrations (5 mg/ml) in the growth medium, lysis of stationary phase yeast cells was observed.