Purification and identification of barley (Hordeum vulgare L.) proteins that inhibit the alkaline serine proteinases of Fusarium culmorum

It has been proposed that microbial proteinase inhibitors, which are present in abundance in cereal grains, protect the seed against plant pathogens. So far, however, very little is known about the interactions of those inhibitors with the proteinases of phytopathogenic microbes. The increased alkaline proteinase activities of Fusarium head blight (FHB) diseased wheat and barley grain imply that the Fusarium fungi synthesize those enzymes during the colonization of the kernel. To study which barley proteins can inhibit Fusarium proteinases, and hence, possibly protect the seed from FHB, the proteins of a grain extract have been separated and tested for their abilities to inhibit two alkaline serine proteinases that we previously isolated from F. culmorum. The proteins were separated by size exclusion, ion exchange, and reversed-phase-HPLC chromatographies. The purified inhibitors were identified by their molecular masses and N-terminal amino acid sequences. The proteins that inhibited the subtilisin-like Fusarium proteinase were the chymotrypsin/subtilisin (CI) inhibitors 1A, 1B, and 2A and the barley alpha-amylase/subtilisin inhibitor (BASI). Only one of the purified proteins inhibited the trypsin-like proteinase, the barley Bowman-Birk inhibitor (BBBI). No novel inhibitors were detected.     

Kinetic study of the activation process of a latent mushroom (Agaricus bisporus) tyrosinase by serine proteases.

Latent mushroom tyrosinase can be considered as a zymogen when activated by proteases because the activation process fulfilled all of the kinetic dependencies predicted by a theoretical zymogen activation model previously reported. The activation was studied under two assay conditions: high and low ratio of latent tyrosinase/serine protease (trypsin and subtilisin Carlsberg) concentrations, in the presence and in the absence of a serine protease inhibitor (aprotinin). The size of the latent enzyme was 67 kDa, determined by denaturing SDS-PAGE electrophoresis and Western blot assays. After proteolytic activation, the size was 43 kDa, with an intermediate band of 58 kDa. The values of the catalytic () and Michaelis () constants for the active forms of tyrosinase resulting from the activation by subtilisin, trypsin, or sodium dodecyl sulfate on the substrate tert-butylcatechol were slightly different, which could support the idea of "one activator-one different active tyrosinase". Vacuum infiltration experiments tried to reproduce in vivo the role of mushroom serine proteases in the activation of latent tyrosinase. The use of serine protease inhibitors is proposed as a new alternative tool to prevent melanin formation.     

Reaction of lentil trypsin-chymotrypsin inhibitors with human and bovine proteinases

Four trypsin-chymotrypsin inhibitors from Syrian local small lentils were selected to study the reasons for their different action against human and bovine proteinases. Chemical modification experiments, enzymatic modifications followed by carboxypeptidase degradation, and characterization of the inhibitor/enzyme complexes formed were performed. All four Lens culinaris inhibitors (LCI) contained arginine at the trypsin-reactive site, and tyrosine (LCI-1.7 and LCI-2.2), phenylalanine (LCI-3.3), or leucine (LCI-4.6) at the chymotrypsin-reactive site. The inhibition of more than one molecule of human chymotrypsin per molecule of inhibitor was caused by the additional and atypical binding at the trypsin-reactive site of all four inhibitors. The approximately 2.5-fold higher inhibition of human chymotrypsin compared to bovine chymotrypsin was the result of two effects, the additional binding of human chymotrypsin at the trypsin-reactive site and the low inhibition of bovine chymotrypsin. As a consequence, human enzyme preparations or suitable conversion factors should be used to evaluate the effect of such inhibitors in foods.     

Multienzyme inhibition assay for residue analysis of insecticidal organophosphates and carbamates

A recently developed spectrophotometric assay for the detection of organophosphorus and carbamate insecticides by means of cutinase inhibition has been successfully extended to two esterases derived from Bacillus subtilis (BS2) and rabbit liver. These esterases were selected because of their high sensitivity to the examined insecticide classes and their pronounced inhibition profile. With inhibition constants (ki) of 2.0x10(7) and 2.6x10(6) L/(mol.min) for rabbit liver esterase and BS2, respectively, chlorpyrifos oxon proved to be the strongest inhibitor directly followed by paraoxon. As compared to choline esterases and the recently studied cutinase, both esterases are surprisingly strongly inhibited by organophosphorus thions, showing k i in the range of 5.3x10(2) to 2.3x10(4) L/(mol.min). All tested insecticidal carbamates were also inhibitors of BS2 and rabbit liver esterase, albeit in a rather uniform manner. Generally, both enzymes were found to be about 2 orders of magnitude more sensitive on the studied insecticides than cutinase even with an enhanced sensitivity against plant matrix effects. Plant extracts, obtained according to the QuEChERS method, were subjected to solid-phase extraction (SPE) using a mixed mode strong anion exchanger/primary secondary amine sorbent and C18endcapped cartridges for superior cleanup. With spiked samples of apple juice, best recoveries of 73% (+/-61%), 94% (+/-25%), and 134% (+/-17%) were obtained for chlorpyrifos, parathion-methyl, and paraoxon, respectively. Results of exemplarily performed liquid chromatography-mass spectrometry control measurements were well in accordance with measurements obtained by enzyme inhibition.     

Primary structure of the abundant seed albumin of Theobroma cacao by mass spectrometry

The most abundant albumin present in seeds of Theobroma cacao was purified to apparent homogeneity as judged by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH(2)-terminal sequence analysis. Tryptic peptide mass fingerprinting of the purified protein by HPLC/ESI-MS showed the presence of 16 masses that matched the expected tryptic peptides corresponding to 95% of the translated amino acid sequence from the cDNA of the 21 kDa cocoa albumin. Collision-induced dissociation MS/MS analysis of the C-terminal peptide isolated from the CNBr cleavage products provided unequivocal evidence that the mature cocoa albumin protein is nine amino acid residues shorter than expected from the reported cDNA of its corresponding gene. The experimentally determined M(r) value of 20234 was in excellent agreement with the truncated version of the amino acid sequence. The purified cocoa albumin inhibited the catalytic activities of bovine trypsin and chymotrypsin. The inhibition was stoichiometric with 1 mol of trypsin or chymotrypsin being inhibited by 1 mol of inhibitor with apparent dissociation constants (K(i)) of 9.5 x 10(-8) and 2. 3 x 10(-6) M, respectively, for inhibitor binding at pH 8.5 and 37 degrees C. No inhibition of the catalytic activities of subtilisin, papain, pepsin, and cocoa endoproteases was detected under their optimal reaction conditions.     

Evaluation of the selective respiratory inhibition method for measuring the ratio of fungal:bacterial activity in acid agricultural soils

A procedure for the measurement of the fungal and bacterial contribution to substrate-induced respiration was tested in three arable soils. Glucose and different amounts of cycloheximide (eukaryote inhibitor) and streptomycin sulfate (prokaryote inhibitor) were added to soil suspensions, and respiration (CO₂ evolution) was measured. Streptomycin sulfate concentrations from 10 to 120 mg ml^–1 soil solution caused a stable inhibition of respiration. Amounts of cycloheximide ranging from 5 to 35 mg ml^–1 showed an increasing inhibition. In a test with separate and combined addition of the antibiotics at maximum inhibitory concentrations, inhibition by streptomycin was completely overlapped by cycloheximide. This indicated non-target inhibition which may lead to overestimation of fungal respiration. Experiments with sterilized soils inoculated with either fungi or bacteria confirmed that streptomycin selectively inhibited bacteria. Cycloheximide, however, did not only inhibit fungal respiration already at 2 mg ml^–1, but also increasingly inhibited bacterial respiration at increasing concentrations. Only at less than 5 mg cycloheximide ml^–1 was the condition of selective fungal inhibition fulfilled. When 2 mg cycloheximide and 10 mg streptomycin sulfate ml^–1 were applied, the sum of the separate inhibitions almost equalled the combined inhibition by the mix of both inhibitors in field samples. This method yielded fungal:bacterial respiration ratios of 0.50 to 0.60, and confirmed the dominance of bacteria in Dutch arable soils. The ratios obtained by the selective inhibitors were not correlated with, and were higher than, ratios of fungal:bacterial biovolume (0.19 to 0.46) as determined by microscopy and image analysis. Similar measurements in a forest soil (A-horizon) raised doubts on the reliability of the fungal inhibition by cycloheximide in this soil. It is concluded that the separate:combined inhibition ratio should always be checked, and comparison with other approaches is recommended. Received: 17 September 1996     

钾解吸动力学方程及方程常数的应用

Elovich,parabolic diffusion,power function and exponential equations were used to describe K desorption kinetics of 12 soils in a constant electric field of electro-ultrafiltration(EUF),Results showed that the Elovich,parabolic diffusion and power function equations could describe K desorption kinetics well owing to their high correlation coefficients and low standard errors;but the exponential equation was not suitable to be used in this study due to its relatively low correlation coefficients and relatively high standard errors.This work established successfully the relationships between the constants(slope or intercept)of kinetic equations and the barley responses to K fertilizer in the multiple-site field experiments and K-supplying status of soilsk,the constants of Elovich,parabolic diffusion and power function equations were very significantly or significantly correlated to the soil available K,relative yield of barley and K uptake of barley in NP plot.It was suggested that the kinetic equation constants could be used to estimate K-supplying power of soils.     

Rapid gastric fluid digestion and biochemical characterization of engineered proteins enriched in essential amino acids.

The barley high lysine (BHL) proteins are nutritionally enhanced derivatives of barley chymotrypsin inhibitor-2 (CI-2). A compactly folded new CI-2 derivative, BHL9, was engineered with the highest content of threonine, tryptophan, and isoleucine yet achieved in this protein family (15.1, 9.4, and 12.1 wt %, respectively). BHL9 had an unfolding midpoint of 5.5 M guanidinium chloride, significantly greater than values for wild type (3.9 M) or for the previously most stable BHL protein, BHL8 (3.6 M). BHL9 and all other derivatives were digested within 15 s in simulated gastric fluid (SGF), suggesting nutritional availability upon ingestion. Denaturation of the proteins in SGF minus pepsin was revealed by changes in their fluorescence emission spectra and/or far UV circular dichroism spectra. The proteins lack homology to known allergens. Significantly, the BHL8 and BHL9 proteins were stable to proteases at pH 7.5 or 8.0, attesting to their potential for high expression in plants.     

Isolation of a new phlorotannin, a potent inhibitor of carbohydrate-hydrolyzing enzymes, from the brown alga Sargassum patens

Ethanol extracts from 15 kinds of marine algae collected from the coast of the Noto Peninsula in Japan were examined for their inhibitory effects on human salivary α-amylase. Four extracts significantly suppressed the enzyme activity. An inhibitor was purified from the extract of Sargassum patens . The compound was a new phloroglucinol derivative, 2-(4-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy) benzene-1,3,5-triol (DDBT), which strongly suppressed the hydrolysis of amylopectin by human salivary and pancreatic α-amylases. The 50% inhibitory activity (IC(50)) for α-amylase inhibition of DDBT (3.2 μg/mL) was much lower than that of commercially available α-amylase inhibitors, acarbose (26.3 μg/mL), quercetagetin (764 μg/mL), and α-amylase inhibitor from Triticum aestivum (88.3 μg/mL). A kinetic study indicated that DDBT was a competitive α-amylase inhibitor with a K(i) of 1.8 μg/mL. DDBT also inhibited rat intestinal α-glucosidase with an IC(50) value of 25.4 μg/mL for sucrase activity and 114 μg/mL for maltase activity. These results suggest that DDBT, a potent inhibitor of carbohydrate-hydrolyzing enzymes, may be useful as a natural nutraceutical to prevent diabetes.     

Effect of soil on urease inhibition by substituted urea herbicides

The effects of some substituted urea herbicides, fenuron, monuron, diuron and linuron, on soil urease were investigated. All herbicides are soil urease mixed inhibitors and the same inhibition mechanism is presumed. A kinetic relationship, which takes into account herbicide adsorption, is developed in order to calculate the inhibition constants of soil urease from adsorption constants. A linear relationship between Hammett sigma values and log K_i for fenuron, monuron and diuron is obtained, from which the formation of a complex between herbicides and enzyme is proposed By comparing kinetic constants for soil urease with those obtained for jack bean, in the presence of the same herbicides, a possible effect of the soil matrix on the enzyme-herbicide complex is also suggested.     

Slow-binding inhibition of mushroom (Agaricus bisporus) tyrosinase isoforms by tropolone.

A kinetic study of the inhibition of mushroom tyrosinase by tropolone has been made. Three tyrosinase isoforms were used: two commercial tyrosinases from Fluka and Sigma (isoelectric points of 4. 3 and 4.1, respectively) and one purified isoform from mushroom strain U1 (isoelectric point of 4.5). Tropolone is a slow-binding inhibitor of these mushroom tyrosinase isoforms. Increasing tropolone concentrations provoked a progressive decrease in both the initial velocity and the final (inhibited) steady-state rate in the progress curves of product accumulation. A rapid formation of an enzyme-inhibitor complex, which further undergoes a slow reversible reaction, could take place since the inhibition of the different isoforms was partially reversed by the addition of CuSO(4). The kinetic parameters that described the inhibition by tropolone were evaluated by nonlinear regression fits. Incubation experiments of the different isoforms with tropolone demonstrated that this inhibitor only could bind to the "oxy" form of tyrosinase which justifies a mechanism previously proposed to explain the inhibition of tyrosinase by slow-binding inhibitors.     

Vanadate inhibition of fungal PhyA and bacterial AppA2 histidine acid phosphatases

The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shut down by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading activities of fungal PhyA phytase and bacterial AppA2 phytase, kinetic experiments were performed in the presence and absence of orthovanadate and metavanadate under various acidic pHs. Orthovanadate was found to be a potent inhibitor at pH 2.5 to 3.0. A 50% activity of fungal phytase was inhibited at 0.56 μM by orthovanadate. However, metavanadate preferentially inhibited the bacterial AppA2 phytase (50% inhibition at 8 μM) over the fungal phytase (50% inhibition at 40 μM). While in bacterial phytase the K(m) was not affected by ortho- or metavanadate, the V(max) was reduced. In fungal phytase, both the K(m) and V(max) was lowered. The vanadate exists as an anion at pH 3.0 and possibly binds to the active center of phytases that has a cluster of positively charged Arg, Lys, and His residues below the enzymes' isoelectric point (pI). The active site fold of haloperoxidase was shown to be very similar to fungal phytase. The vanadate anions binding to cationic residues in the active site at acidic pH thus serve as a molecular switch to turn off phytase activity while turning on the haloperoxidase activity. The fungal PhyA phytase's active site housing two distinct reactive centers, one for phosphomonoesterase and the other for haloperoxidase, is a unique example of how one protein could catalyze two dissimilar reactions controlled by vanadate.     

Kinetic solvent effects on phenolic antioxidants determined by spectrophotometric measurements

The effects of polar (acetonitrile and tert-butyl alcohol) and apolar (cyclohexane) solvents on the peroxyl-radical-trapping antioxidant activity of some flavonoids, catechol derivatives, hydroquinone, and monophenols have been studied. The inhibition rate constants k(inh) of the antioxidants have been determined by following the increase in absorbance at 234 nm of a dilute solution of linoleic acid at 50 degrees C containing small amounts of antioxidant and radical initiator. Despite the low concentration of linoleic acid, the peroxidation process has been confirmed to be a free radical chain reaction described by the classical kinetic laws for this process. However, in the evaluation of k(inh), a careful analysis of the peroxidation curve, absorbance versus time, must be done because the final oxidation products of phenols may absorb at 234 nm. Phenols with two ortho-hydroxyls are the most active antioxidants, with inhibition rate constants in the range of (3-15) x 10(5) M(-1) x s(-1) (in cyclohexane). Nevertheless, it has been observed that in tert-butyl alcohol (a strong hydrogen bond acceptor) the rate constants dramatically decline to values not detectable by the present kinetic method. In acetonitrile (a weaker hydrogen bond acceptor) instead, the phenols with two ortho-hydroxyls scavenge the peroxyl radicals with rate constants close to those in cyclohexane. From the kinetic solvent effect, the equilibrium constant of the first solvation step of hydroquinone with tert-butyl alcohol has been determined at 50 degrees C, K(1) = 2.5 +/- 0.5 M(-1).     

Study of metallopeptidase isozymes from malted barley (Hordeum vulgare cv. Morex)

It has been reported that germinated barley contains peptidases that are sensitive to metal-chelating agents; however, none of these enzymes have been isolated, nor have their roles in germinated barley been investigated. Anion-exchange chromatography and chromatofocusing have been used to isolate a group of peptidases from barley (Hordeum vulgare cv. Morex) green malt that are sensitive to metal-chelating agents. Their activities were studied using one- and two-dimensional polyacrylamide gel electrophoresis. When analyzed on two-dimensional PAGE gels that contained gelatin as substrate, the enzymes separated into three major and approximately six minor activity spots with acidic pI values. The enzymes were optimally active against the gelatin substrate at pH 8.0 and were completely inhibited by 1,10-phenanthroline and EDTA, indicating that they belonged to the metallopeptidase class (EC 3.4.24.x). After the enzymes were inhibited with EDTA, the activities were recovered in the presence of low concentrations of metal ions. The hydrolysis of gelatin substrate was also impaired by the presence of reducing agents. The metallopeptidases readily digested, in vitro, the barley prolamine D hordein, indicating that they may be involved in degrading storage proteins during barley germination.     

Kinetics of K Desorption from Soils in a Constant Electric Field (Electro-Ultrafiltration) and Its Application

Kdesorption from soils in a constant electric field (field strength:44.5Vcm^-1) by means of electro-ultrafil-tration (EUF) followed second-order kinetics and could be described by the equation dd/dt=k(D-d)2. From theequation, such kinetic parameters relating to K desorption from soils as the maximum desorbable quantity D, quantity of K desorbed within 40 minutes d40, initial desorption rate Vo, desorption rate constant k and half-time t1/2 could be calculated. An expression which describes the relationships between the kinetic parameters on the one hand and the responses of barley to fertilizer-K in the field experiments in different sites and the potassium-supplying power of soils on the other was established. Vo, D and d40 were significantly correlated with barley relative yield, K uptake by barley and the content of soil available potassium. The rate constants of K desorption varied between 4.42×10^-4-1.80×10^-3kg mg^-1 min^-1 and highly correlated with the relative yield of barley.     

Irreversible competitive inhibitory kinetics of cardol triene on mushroom tyrosinase

Cardol triene was first purified from cashew (Anacardium occidentale L.) nut shell liquid and identified by gas chromatography coupled to mass spectroscopy and nuclear magnetic resonance. The effects of this compound on the activity of mushroom tyrosinase were studied. The results of the kinetic study showed that cardol triene was a potent irreversible competitive inhibitor and the inactivation was of the complexing type. Two molecules of cardol triene could bind to one molecule of tyrosinase and lead to the complete loss of its catalytic activity. The microscopic rate constants were determined for the reaction of cardol triene with the enzyme. The anti-tyrosinase kinetic research of this study provides a comprehensive understanding of inhibitory mechanisms of resorcinolic lipids and is beneficial for the future design of novel tyrosinase inhibitors.     

Identification of a Kunitz-type proteinase inhibitor from Pithecellobium dumosum seeds with insecticidal properties and double activity

A trypsin inhibitor, PdKI, was purified from Pithecellobium dumosum seeds by TCA precipitation, trypsin-sepharose chromatography, and reversed-phase-HPLC. PdKI was purified 217.6-fold and recovered 4.7%. SDS-PAGE showed that PdKI is a single polypeptide chain of 18.9 kDa and 19.7 kDa by MALDI-TOF. The inhibition on trypsin was stable in the pH range 2-10 and at a temperature of 50 degrees C. The Ki values were 3.56 x 10(-8)and 7.61 x 10(-7) M with competitive and noncompetitive inhibition mechanisms for trypsin and papain, respectively. The N-terminal sequence identified with members of Kunitz-type inhibitors from the Mimosoideae and Caesalpinoideae subfamilies. PdKI was effective against digestive proteinase from Zabrotes subfasciatus, Ceratitis capitata, Plodia interpunctella, Alabama argillaceae, and Callosobruchus maculatus, with 69, 66, 44, 38, and 29% inhibition, respectively. Results support that PdKI is a member of the Kunitz inhibitor family and its insecticidal properties indicate a potent insect antifeedant.     

Potential fungal inhibition by immobilized hydrolytic enzymes from Trichoderma asperellum

The use of cell wall degrading enzymes from Trichoderma asperellum immobilized on biodegradable support is an alternative for food packaging. In this study, hydrolytic enzymes produced by T. asperellum were tested as a fungal growth inhibitor, in free form or immobilized on a biodegradable film composed of cassava starch and poly(butylene adipate-co-terephtalate) (PBAT). The inhibitory activity was tested against Aspergillus niger , Penicillium sp., and Sclerotinia sclerotiorum , microorganisms that frequently degrade food packaging. The use of chitin as carbon source in liquid medium induced T. asperellun to produce N-acetylglucosaminidase, β-1,3-glucanase, chitinase, and protease. The presence of T. asperellun cell wall degradating enzymes (T-CWD) immobilized by adsorption or covalent attachment resulted in effective inhibition of fungal growth. The enzymatic activity of T-CWD was stronger on S. sclerotiorum than on the Aspergillus or Penicillum isolates tested. These results suggest that T-CWD can be used in a free or immobilized form to suppress fungi that degrade food packaging.     

Effect of organic matter and urease inhibitors on urea hydrolysis and immobilization of urea nitrogen in an alkaline soil

Summary Laboratory incubation experiments with ¹⁵N-labelled urea were conducted on a Aquic Udifluvent Belgian soil amended with barley straw, in, order to study the influence of three urease inhibitors, hydroquinone, phenyl phosphorodiamidate and N-(n-butyl) phosphorothioic triamide on urea hydrolysis and N transformations. The results demonstrated that the urea was hydrolyzed more rapidly when the soil was amended with ground barley straw. A pronounced inhibition of urease inhibitors occurred with the urea hydrolysis, but it was decreased by increasing the soil organic C content. A severe N immobilization (about 82–100% of the applied urea) occurred in soil samples that were rich in organic C. The addition of urease inhibitors increased urea-N immobilization by 5–30%. N-(n-butyl) phosphorothioic triamide had a stronger effect than the other two inhibitors when they were applied at the rate of 1%. However, the inhibitors decreased N immobilization when the soil was amended with barley straw.     

TAXI type endoxylanase inhibitors in different cereals

An affinity-based purification procedure with the immobilized family 11 Bacillus subtilis endoxylanase XynA allowed us to obtain high yields of highly pure endoxylanase inhibitor fractions from rye, barley, and durum wheat. In contrast, no inhibitors interacting with the B. subtilis endoxylanase affinity column are present in corn, buckwheat, rice, and oats. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and inhibitor specificity showed that the isolated inhibitors belonged to the TAXI endoxylanase inhibitor family, thus providing a view on the diversity of this cereal inhibitor family. The isolated inhibitors are basic proteins of ca. 40 kDa, occurring in two molecular forms, with pI values of ca. 8.5 (durum wheat) and ca. 9.0 (rye, barley). They are, in general, strong inhibitors of family 11 endoxylanases but not of family 10 endoxylanases. Because cereal endogenous endoxylanases belong to the latter family, this probably indicates that they do not influence cereal metabolic processes. For the first time, endoxylanase inhibitors, similar to TAXI I and TAXI II from wheat, were isolated from durum wheat and characterized. For each cereal, high-resolution cation exchange chromatography revealed the presence of multiple isoinhibitors, each of which occurs in two molecular forms. However, in durum wheat and barley, a single isoform is predominantly present.