Antifungal and antimycotoxigenic metabolites in Anacardiaceae species from northwest Argentina: isolation, identification and potential for control of Fusarium species.

AIMS: The purpose of this research was to identify antifungal compounds from leaves of Schinus and Schinopsis species useful for the control of toxigenic Fusarium species responsible of ear rot diseases. METHODS AND RESULTS: Leaves of Schinopsis (S. lorentzii and S. haenkeana) and Schinus (S. areira, S. gracilipes and S. fasciculatus) were sequentially extracted with dichloromethane, ethyl acetate and methanol. The antifungal activity of the fraction soluble in methanol of these extracts (fCH2Cl2, fAcEt and fMeOH, respectively) was determined by the broth microdilution method and the disc-diffusion method. The minimum inhibitory dose (MID), the diameter of growth inhibition (DGI) and the minimum concentration for 50% inhibition of fungal growth (MIC50) were calculated. The fCH2Cl2 and fAcEt of the Schinopsis species had the lowest MID and MIC50 values and the highest DGI. The antifungal compounds were identified as lupeol and a mix of phenolic lipids. The last one had the highest antifungal activity with MIC50 31-28 µg g(-1) and 165-150 µg g(-1) on Fusarium graminearum and Fusarium verticillioides, respectively. The identified metabolites completely inhibited fumonisin and deoxynivalenol production at lower concentrations than ferulic acid, a natural antimycotoxigenic compound. CONCLUSIONS: It was proven that lupeol and phenolic lipids were inhibitors of both fungal growth and mycotoxin production of toxigenic Fusarium species. This fact is specially interesting in the control of the toxigenic Fusarium species because several commercial antifungals showed to stimulate mycotoxin biosynthesis at sublethal concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: Control of toxigenic Fusarium species requires compounds able to inhibit both fungal growth and mycotoxin production. Our results suggest that the use of lupeol as food preservative and the phenolic lipids as fungal growth inhibitors of F. verticillioides and F. graminearum did not imply an increase in mycotoxin accumulation.

Toxigenic potential of Fusarium graminearum isolated from maize of northwest Argentina.

Twenty six isolates of Fusarium graminearum from grains of maize hybrids harvested in ±west Argentina were grown on autoclaved rice grain to assess their ability to produce type B trichothecenes. Chemical analysis indicated that 38% of isolates were nivalenol (NIV) producers only, 31% were major NIV producers with high DON(deoxynivalenol)/NIV ratios, 8% were major DON producers with minor NIV production, and 23% were DON producers only. Isolates showed a high variability in their toxigenic potential which was not related to fungal biomass. The distribution of the different chemotypes as well as the high and the low trichothecene-producing Fusarium isolates could not be associated to a geographical origin. Our results confirmed for the first time that isolates of Fusarium graminearum from maize of northwest Argentina are able to produce DON and NIV. A substancial contamination with both NIV and DON is likely in maize from northwest Argentina. Their contents should be quantified in regional surveillances for mycotoxin contamination.

Development and validation of a micromethod for fast quantification of 5-n-alkylresorcinols in grains and whole grain products.

A 96-well plate micromethod was developed to measure 5-n-alkylresorcinols (5nARs) in cereal grains and food derived products. The 5nARs reacted in alkaline alcoholic medium with Fast Blue RR ½ZnCl2 salt to yield coloured azo-derivatives. The highest sensitivity for 5nARs was obtained at 490 nm with 0.025% ethanolic Fast Blue RR and 5% K2CO3. This reaction showed good linearity for olivetol (0.05-0.20 µg). Contents of 5nARs determined in cereal grains and derived products by the new Fast Blue RR micromethod were highly correlated (R(2)=0.9944) with those obtained by a Fast Blue B method currently used. A Bland-Altman analysis indicated a small positive bias near to zero (R(2)=0.0401), suggesting that the methods can be interchangeably used. The new reaction is completed in 15 min and the coloured products are read within the 15 min after completion. The micromethod offers a fast analysis of 5nARs in cereal grains and derived products with low consumption of reagents and solvents.

Toxigenic potential of Fusarium graminearum isolated from maize of northwest Argentina

Twenty six isolates of Fusarium graminearum from grains of maize hybrids harvested in ±west Argentina were grown on autoclaved rice grain to assess their ability to produce type B trichothecenes. Chemical analysis indicated that 38% of isolates were nivalenol (NIV) producers only, 31% were major NIV producers with high DON(deoxynivalenol)/NIV ratios, 8% were major DON producers with minor NIV production, and 23% were DON producers only. Isolates showed a high variability in their toxigenic potential which was not related to fungal biomass. The distribution of the different chemotypes as well as the high and the low trichothecene-producing Fusarium isolates could not be associated to a geographical origin. Our results confirmed for the first time that isolates of Fusarium graminearum from maize of northwest Argentina are able to produce DON and NIV. A substancial contamination with both NIV and DON is likely in maize from northwest Argentina. Their contents should be quantified in regional surveillances for mycotoxin contamination.

Toxigenic potential of Fusarium graminearum isolated from maize of northwest Argentina

Twenty six isolates of Fusarium graminearum from grains of maize hybrids harvested in ±west Argentina were grown on autoclaved rice grain to assess their ability to produce type B trichothecenes. Chemical analysis indicated that 38% of isolates were nivalenol (NIV) producers only, 31% were major NIV producers with high DON(deoxynivalenol)/NIV ratios, 8% were major DON producers with minor NIV production, and 23% were DON producers only. Isolates showed a high variability in their toxigenic potential which was not related to fungal biomass. The distribution of the different chemotypes as well as the high and the low trichothecene-producing Fusarium isolates could not be associated to a geographical origin. Our results confirmed for the first time that isolates of Fusarium graminearum from maize of northwest Argentina are able to produce DON and NIV. A substancial contamination with both NIV and DON is likely in maize from northwest Argentina. Their contents should be quantified in regional surveillances for mycotoxin contamination.(AU)

Species diversity and toxigenic potential of Fusarium graminearum complex isolates from maize fields in northwest Argentina.

Members of the Fusarium graminearum species complex (Fg complex) are the causal agents of ear rot in maize and Fusarium head blight of wheat and other small grain cereals. The potential of these pathogens to contaminate cereals with trichothecene mycotoxins is a health risk for both humans and animals. A survey of ear rot isolates from maize collected in northwest Argentina recovered 66 isolates belonging to the Fg complex. A multilocus genotyping (MLGT) assay for determination of Fg complex species and trichothecene chemotypes was used to identify 56 of these isolates as F. meridionale and 10 isolates as F. boothii. F. meridionale was fixed for the nivalenol (NIV) chemotype, and all of the F. boothii isolates had the 15-acetyldeoxynivalenol (15ADON) chemotype. The results of genetic diversity analysis based on nine variable number tandem repeat (VNTR) loci supported the hypothesis of genetic isolation between F. meridionale and F. boothii, and provided little evidence of geographic substructure among populations of the dominant pathogen species, F. meridionale. This is the first study to indicate that F. meridionale and F. boothii may play a substantial role in the infection and trichothecene contamination of maize in Argentina. In addition, dominance of the NIV chemotype among Fg complex isolates from Argentina is unprecedented, and of significant concern to food safety and animal production.

A molecular based strategy for rapid diagnosis of toxigenic Fusarium species associated to cereal grains from Argentina.

Fusarium species are worldwide causal agents of ear rot in cereals. Their toxigenic potential is a health risk for both humans and animals. In Argentina, most identification of these fungi has been based on morphological and cross-fertility criteria which are time consuming and require considerable expertise in Fusarium taxonomy and physiology. DNA based approaches have been reported as rapid, sensitive and specific alternatives to identify the main fumonisin and trichothecene-producing Fusarium species. In this work, we used PCR assays and the partial sequence of TEF1-alpha gene (Translation Elongation Factor-1 alpha) to identify the fumonisin and trichothecene-producing species in Fusarium isolates from diverse regions of Argentina. The relative efficiency and reliability of those methods to improve mycotoxin risk prediction in this country were also assessed. Species-specific PCR assays were targeted toward multicopy IGS (Intergenic Spacer of rDNA units) and on the toxin biosynthetic genes FUM1 (fumonisins) and TRI13 and TRI7 genes (trichothecenes). PCR assays based on FUM1 gene and IGS sequences allowed detection and discrimination of the fumonisin producers Fusarium proliferatum and Fusarium verticillioides. Molecular identification of nonfumonisin producers from Gibberella fujikuroi species complex was possible after determination of TEF1-alplha gene sequences, which indicated the presence of Fusarium subglutinans, Fusarium andiyazi and Fusarium thapsinum. TEF-1 alpha gene sequences also allowed discrimination of the different species of the Fusarium graminearum complex (F. graminearum sensu lato) as F. graminearum sensu stricto, Fusarium meridionale and Fusarium boothii. The last two species belonged to NIV chemotype and were detected for the first time in the subtropical region of Argentina while F. graminearum sensu stricto was DON producer only, which was also confirmed by specific PCR assays based on TRI137/TRI7 genes. Our results indicated that the PCR assays evaluated in this work are reliable diagnostic tools to detect the main toxigenic Fusarium species associated to cereal grains in Argentina. An extensive epidemiological survey based on the approach presented in this work is currently in progress to know the mycotoxigenic hazard of Fusarium species in cereal grains from the subtropical region of Argentina.

Purification and identification of antibacterial phenolics from Tripodanthus acutifolius leaves.

AIMS: To perform an activity-guided purification, identification and quantification of antibacterial compounds from Tripodanthus acutifolius infusion. To validate the antibacterial activity of purified substances. METHODS AND RESULTS: Bioautographic methods were employed as screening assays for purifying bioactive substances. Purification procedures included sephadex LH-20 column chromatography and reverse phase HPLC. Identification was achieved by spectroscopic methods (UV-Vis, MS, NMR and polarimetry) and chromatographic assays (paper chromatography and HPLC). Antibacterial activity was studied by microdilution, colony count and photometric assays, Sytox green stain and transmission electron microscopy (TEM). Four glycoflavonoids (rutin, nicotiflorin, hyperoside and isoquercitrin) and an unusual phenylbutanoid glycoside (tripodantoside) were purified and identified. Tripodantoside was found at 6.59 +/- 0.82 g per 100 g of dry leaves. The flavonoids showed bactericidal effect at a concentration of 4 mg ml(-1) against Staphylococcus aureus and Pseudomonas aeruginosa strains from American Type Culture Collection, while tripodantoside was almost four times more active than those compounds, with a minimum bactericidal concentration = 1.024 mg ml(-1) against these strains. Tripodantoside aglycone showed bacteriolytic effects on the assayed strains, causing evident damages on cell wall and membrane, while tripodantoside did not exhibit those effects. CONCLUSIONS: The antibacterial activity of T. acutifolius infusion would be partially attributed to the purified glycoflavonoids and mainly to tripodantoside. SIGNIFICANCE AND IMPACT: The high extraction yield and the antibacterial activity exhibited by tripodantoside makes this chemical structure of interest to support further studies dealing with chemical modifications to increase the antibacterial activity or to seek another activities.

Antibacterial activity of plant extracts from northwestern Argentina.

AIMS: To determine the antibacterial and cytotoxic activities of aqueous and ethanolic extracts of northwestern Argentinian plants used in folk medicine. To compare the mentioned activities with those of five commercial antibiotics. To identify the compounds responsible for the antibacterial activity. METHODS AND RESULTS: Plant extracts were prepared according to traditional uses in northwestern Argentina. Antibacterial activity was assayed by agar dilution in Petri dishes and broth dilution in 96-well plates. Lethal dose 50 (LD(50)) was determined by the Artemia salina assay. Phytochemical analysis was performed by sample adsorption on silica gel, thin-layer chromatography (TLC), bioautography and UV-visible spectra. The results showed that Tripodanthus acutifolius aqueous extracts have lower minimal inhibitory concentrations (MIC) (502 and 506 microg of extracted material (EM) per ml for infusion and decoction, respectively) than cefotaxim MIC (640 microg ml(-1)) against Acinetobacterfreundii (303). These data were lower than their LD(50). Tripodanthus acutifolius tincture showed lower MIC (110 microg of EM per ml) and minimal bactericidal concentration (MBC) (220 microg of EM per ml) than cefotaxim (MIC and MBC of 320 microg ml(-1)) for Pseudomonasaeruginosa. This extract also showed a MIC/MBC of 110/220 microg of EM per ml, lower than oxacillin (MIC/MBC of 160/220 microg ml(-1)) for Staphylococcus aureus (ATCC 25923). The cytotoxicity of all extracts were compared with that of commercial antibiotics. Rutin (3,3',4',5,7-pentahydroxyflavone 3-beta-rhamnosilglucoside), iso-quercitrin (3,3',4',5,7-pentahydroxyflavone 3-beta-glucoside) and a terpene would be partially responsible for the antibacterial activity of T. acutifolius infusion. CONCLUSIONS: Tripodanthus acutifolius extracts had the ability to inhibit bacterial growth. The antibacterial activity differs with the applied extractive method, and it could be partially attributed to glycoflavonoids. This paper contributes to the knowledge of antibacterial capacity of plants from northwestern Argentina. SIGNIFICANCE AND IMPACT OF THE STUDY: These antibacterial activities support further studies to discover new chemical structures that can contribute to alleviate or cure some illnesses.

Propolis from the northwest of Argentina as a source of antifungal principles.

AIMS: To determine the antimycotic and cytotoxic activities of partially purified propolis extract on yeasts, xylophagous and phytopathogenic fungi. To compare these activities with pinocembrin and galangin isolated from this propolis and with the synthetic drugs ketoconazole and clortrimazole. METHODS AND RESULTS: Ethanolic propolis extract was partially purified by cooling at -20 degrees C. Two of its components were isolated by HPLC and identified as pinocembrin and galangin. The antifungal activity was assayed by bioautography, hyphal radial growth, hyphal extent and microdilution in liquid medium. Cytotoxicity was studied with the lethality assay of Artemia salina. The obtained results were compared with the actions of ketoconazole and clortrimazole. The results showed that the antifungal potency of ketoconazole and clortrimazole is higher than pinocembrin, galangin and the partially purified propolis extract in this order. Otherwise, the cytotoxicity of the synthetic drugs is also the highest. CONCLUSIONS: Partially purified propolis extract inhibits fungal growth. The comparison of its relative biocide potency and cytotoxicity with synthetic drugs and two components of this propolis (pinocembrin and galangin) showed that the propolis from 'El Siambón', Tucumán, Argentina, is a suitable source of antifungal products. SIGNIFICANCE AND IMPACT OF THE STUDY: The partially purified propolis extract and its isolated compounds, pinocembrin and galangin, have the capacity of being used as antifungals without detriment to the equilibrium of agroecosystems. The impact of this study is that the preparation of agrochemicals with reduced economic costs using a partially purified preparation as the active principle is possible.

In vitro fungitoxic activity of Larrea divaricata cav. extracts.

AIMS: To evaluate the fungitoxic activity of Larrea divaricata Cav. extract and one of its components against yeasts and fungi. This activity was compared with the action of ketoconazole, a known synthetic antimycotic. METHODS AND RESULTS: Antifungal activity of Larrea divaricata extract and of a fraction (Fr. B) purified by thin layer chromatography, was investigated using different methodologies. Both exhibited strong activity against the majority of the assayed fungi. Only Fusarium oxysporum and Schizophyllum commune growth was not affected with the assayed conditions. The fungitoxic and cytotoxic activity of the ethanolic extract and ketoconazole were compared. CONCLUSIONS: Ethanolic extracts of L. divaricata Cav. produce growth inhibition of several fungi. One of its constituents with the same activity was purified and identified as a glycoside of a flavanone. A comparison with the action of ketoconazole, which is currently used as antimycotic and can cause adverse health effects was made. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that L. divaricata extract contains, at least, one compound of phenolic nature, with fungitoxic potency against yeasts and fungi.

Antibacterial activity of ethanolic and aqueous extracts of Acacia aroma Gill. ex Hook et Arn.

The purpose of the present study was to investigate the antibacterial activity of seven ethanolic extracts and three aqueous extracts from various parts (leaves, stems and flowers) of A. aroma against 163 strains of antibiotic multi-resistant bacteria. The disc diffusion assay was performed to evaluate antibacterial activity of the A. aroma crude extracts, against several Gram-positive bacteria (E. faecalis, S. aureus, coagulase-negative stahylococci, S. pyogenes, S. agalactiae, S. aureus ATCC 29213, E. faecalis ATCC 29212) and Gram-negative bacteria (E. coli., K. pneumoniae, P. mirabilis, E. cloacae, S. marcescens, M morganii, A. baumannii, P. aeruginosa, S. maltophilia, E. coli ATCC 35218, P. aeruginosa ATCC 27853, E. coli ATCC 25922). All ethanolic extracts showed activity against gram-positive bacteria. Among all obtained extracts, only leaf and flower fluid extracts showed activity against Gram-negative bacteria. Based on this bioassay, leaf fluid extracts tended to be the most potent, followed by flower fluid extracts. Minimal inhibitory concentration (MIC) values of extracts and antibiotics were comparatively determined by agar and broth dilution methods. Both extracts were active against S. aureus, coagulase-negative stahylococci, E. faecalis and E. faecium and all tested Gram-negative bacteria with MIC values from 0.067 to 0.308 mg/ml. In this study the minimal bactericidal concentration (MBC) values were identical or twice as high than the corresponding MIC for leaf extracts and four or eight times higher than MIC values for flower extracts. This may indicate a bactericidal effect. Stored extracts have similar antibacterial activity as recently obtained extracts. The A. aroma extracts of leaves and flowers may be useful as antibacterial agents against Gram- negative and Gram-positive antibiotic multi-resistant microorganisms.

Proteinaceous inhibitor versus fructose as modulators of Pteris deflexa invertase activity.

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer Mr 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had beta-fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with Km of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.

An N-acetylglucosamine oligomer binding agglutinin (lectin) from ripe Cyphomandra betacea Sendt. fruits.

A new agglutinin (lectin), called CBL3, was purified from the juice of ripe Cyphomandra betacea Sendt. fruits until electrophoretically pure to homogeneity. The lectin is a homodimer of M(r)=50800 with subunits of 26200 bound by disulfide linkages with a pI of 4.9. The agglutinating capacity of the lectin is only inhibited by oligomers of N-acetylglucosamine in the following order of potency: tetrasaccharide>trisaccharide>disaccharide. CBL3 is not inhibited by N-acetylglucosamine, the same as all known lectins of the Solanaceae family. The human blood group recognition is non-specific. The lectin is a glycoprotein with 13.6% (w/w) of carbohydrates. The agglutinating activity is not affected by EDTA nor by cations. Mitogenic activity was not detected. Heat and pH stability, amino acid composition, N-terminal amino acid sequence and immunological properties show substantial differences to the reported lectins isolated from the Solanaceae family.

Antioxidant activity of Argentine propolis extracts.

Propolis is used in Argentine folk medicine. We have examined its possible protective action against oxidative modification of lipid in unfractionated serum. The kinetics of copper-induced oxidation was continuously monitored by measuring the formation of conjugated dienes, as the increase in the absorbance at 234 nm. According to the kinetics of oxidation, the propolis were classified in three different groups. Group I (CE, CO, BO, MO, BE) inhibited lipid oxidation during the initiation and propagation phases even at low concentrations. Group II (SP, CA, AM) increased the lag-phase for conjugated diene formation. All propolis in groups I and II diminished the maximal rate of diene production and the maximal amount of dienes produced. Group III (PA, RA, FE, VR, TV) had no effect on the lipid oxidation. The extent of lipoprotein oxidation was measured by the thiobarbituric acid reactive substance assay. Generation of malondialdehyde-like substances was inhibited and delayed by the presence of propolis extracts from group I and II. Our results justify the use of propolis (groups I and II) as a source of natural antioxidants.

Screening antifungal activities of selected medicinal plants.

Plants synthesise a vast array of secondary metabolites that are gaining importance for their biotechnological applications. The antifungal activity of the ethanolic extracts of ten Argentinean plants used in native medicine is reported. Antifungal assays included radial growth inhibition, disk and well diffusion assays and growth inhibition by broth dilution tests. The chosen test fungi were yeasts, microfungi and wood-rot causing Basidiomycetes. Extracts of Larrea divaricata, Zuccagnia punctata and Larrea cuneifolia displayed remarkable activity in the assays against the majority of the test fungi. In addition to the former plants, Prosopanche americana also inhibited yeast growth.

An invertase inhibitory protein from Pteris deflexa link fronds.

Plant invertases play important roles in sucrose metabolism. Cell wall invertase was reported to participate in phloem loading and unloading. Soluble invertases would be involved in hexose level regulation in mature tissues and in stored sucrose utilization within vacuoles. Invertase inhibitory proteins were described as one of the possible mechanisms for invertase activity regulation in some plant species; nevertheless, these proteins were found only in sink tissues, suggesting that this mechanism would not be relevant in the sucrose turnover of leaves. This report describes the purification of invertase from Pteris deflexa fronds and the occurrence of an invertase inhibitory protein in this fern organ, as well as its purification and invertase-inhibitor interactions. The Mr of the invertase and of its inhibitory protein were 90,000 and 18,000, respectively. SDS-PAGE in the presence of 2-mercaptoetanol gave two subunits for the enzyme (Mr=66,000 and 30,000) and only one for the inhibitor. The inhibitor protein is a glycoprotein (12% w/w of neutral sugars) that did not show agglutinating activity like some others, and also showed a high heat stability at pH 5.0. The optimum pH of invertase activity is 5.0, while invertase inhibitory protein caused maximal inhibition at the same pH value. Invertase-inhibitor complex formation occurs in an immediate manner and a protease activity was discarded. The inhibition is non-competitive (Ki=1.5 x 10(-6) M) without interactions among the binding sites. The complex is slightly dissociable and sucrose was able to partially reduce the inhibitory effect. Up to the present, invertase inhibitory proteins have been found solely in heterotrophic tissues. In this work we demonstrate that this protein is also present in an autotrophic tissue of a lower vascular plant.

Comparison of the free radical-scavenging activity of propolis from several regions of Argentina.

Propolis is extensively used in Argentine folk medicine. Alcoholic extracts of propolis from different regions of Argentina were prepared. The extracts were analysed for the determination of total flavonoid content (from 13.3 to 42.6 mg/g of propolis) by using the aluminum nitrate method, UV spectrophotometry and thin layer chromatography. All of them contained high total flavonoid content. It was also observed that all samples of ethanolic extracts of propolis showed free radical-scavenging activity in terms of scavenging of the radical DPPH but the highest activities were found for samples from Tucumán and Santiago del Estero. In all cases with 20 microg/ml of soluble principles, the percentage of DPPH degradation was different (Banda Oeste: 67.5%; Verónica: 45%; Forres: 35%; Saenz Peña: 20% and Juan José Castelli: 55%). These results may justify their use as a source of natural antioxidants.

Invertase proteinaceous inhibitor of Cyphomandra betacea Sendt fruits.

This work describes a new invertase proteinaceous inhibitor from Cyphomandra betacea Sendt. (tomate de arbol) fruits. The proteinaceous inhibitor was isolated and purified from a cell wall preparation. The pH stability, kinetics of the inhibition of the C. betacea invertase, inhibition of several higher plant invertases and lectin nature of the inhibitor were studied. The inhibitor structure involves a single polypeptide (Mr = 19000), as shown by gel filtration and SDS-PAGE determinations. N-terminal aminoacid sequence was determined. The properties and some structural features of the inhibitor are compared with the proteinaceous inhibitors from several plant species (Beta vulgaris L., Ipomoea batatas L. and Lycopersicon esculentum Mill.). All these inhibitors share lectinic properties, some common epitopes, some aminoacid sequences and a certain lack of specificity towards invertases of different species, genera and even plant family. In consequence, the inhibitors appear to belong to the same lectin family. It is now known that some lectins are part of the defence mechanism of higher plants against fungi and bacteria and this is a probable role of the proteinaceous inhibitors.

Invertase activity associated with the walls of Solanum tuberosum tubers.

Three fractions with invertase activity (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were isolated from mature Solanum tuberosum tubers: acid soluble invertase, invertase I and invertase II. The first two invertases were purified until electrophoretic homogeneity. They are made by two subunits with an apparent M(r) value of 35,000 and their optimal pH is 4.5. Invertase I was eluted from cell walls with ionic strength while invertase II remained tightly bound to cell walls after this treatment. This invertase was solubilized by enzymatic cell wall degradation (solubilized invertase II). Their K(m)s are 28, 20, 133 and 128 mM for acid soluble invertase, invertase I, invertase II and solubilized invertase II, respectively. Glucose is a non-competitive inhibitor of invertase activities and fructose produces a two site competitive inhibition with interaction between the sites. Bovine serum albumin produces activation of the acid soluble invertase and invertase I while a similar inhibition by lectins and endogenous proteinaceous inhibitor from mature S. tuberosum tubers was found. Invertase II (tightly bound to the cell walls) shows a different inhibition pattern. The test for reassociation of the acid soluble invertase or invertase I on cell wall, free of invertase activity, caused the reappearance of all invertase forms with their respective solubilization characteristics and molecular and kinetic properties. The invertase elution pattern, the recovery of cell wall firmly bound invertase and the coincidence in the immunological recognition, suggest that all three invertases may be originated from the same enzyme. The difference in some properties of invertase II and solubilized invertase II from the other two enzymes would be a consequence of the enzyme microenvironment in the cell wall or the result of its wall binding.