Soybean Trypsin Inhibitor Research Articles

Contribution of Cathepsin L to Autolysis of Sea Cucumber Stichopus japonicus Intestines

ABSTRACTIn order to provide the fundamental basis for control and application of sea cucumber autolysis, the contribution of cathepsin L to intestinal proteolysis in sea cucumber (Stichopus japonicus) was investigated. Cathepsin L mRNA expression was significantly up-regulated by 1.80 ± 0.44-fold with ultraviolet irradiation (p< 0.05). According to the protein pattern identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and determination of the release of trichloroacetic acid (TCA)-soluble oligopeptide, high intestinal protein degradation was observed at pH 4.4 and 48°C. Intestinal protein proteolysis was strongly inhibited by cathepsin L inhibitors, including trans-epoxysuccinyl-L-leucyl-amido (4-guani-dino)butane (E-64), iodoacetic acid (IA), and antipain (AP). These results imply that cathepsin L is involved in intestinal autolysis in the sea cucumber S. japonicus.Abbreviations: TCA: trichloroacetic acid; Z-Phe-Arg-AMC: Z-Phe-Arg-7-amido-4-methylcou-marin; IA: iodoacetic acid; E-64: trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane; AP: antipain; SBTI: soybean trypsin inhibitor; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; RT-PCR: reverse transcription-polymerase chain reaction

Inhibitory Effect of Protease Inhibitors on Larval Midgut Protease Activities and the Performance of Plutella xylostella (Lepidoptera: Plutellidae).

Plutella xylostella L. (diamondback moth) is a pest of cruciferous plants. To understand the relationship among protease inhibitors, protease activities and the growth and development of this insect, the activities of midgut proteases of P. xylostella larvae were determined in this study. Protease samples were extracted from the midguts of P. xylostella larvae, and the protease activities were determined using enzyme specific substrates. The results showed that CaCl2, EDTA, and EGTA inhibited only the trypsin. Among the common protease inhibitors, phenylmethyl sulfonyl fluorine (PMSF), Nα-p-methyl sulfonyl-L-lysine chloromethylketone (TLCK), Nα-methyl sulfonyl-L- phenylalanine chloromethyl ketone (TPCK), soybean trypsin inhibitor (STI), and PMSF inhibited the total protease, high-alkaline trypsin (a trypsin subtype with highly alkaline pH optimum), low-alkaline trypsin (another trypsin subtype with slightly alkaline pH optimum), and chymotrypsin; TLCK inhibited the total protease and high-alkaline trypsin, whereas TPCK only activated the high-alkaline trypsin activities. STI had an inhibitory effect on all the proteases. These results showed that protease inhibitors had a certain extent inhibition to protease activities in the larval midgut of P. xylostella and that STI can potentially be used for effective pest control. The development of P. xylostella was delayed in the presence of different inhibitors. These effects were also related to the concentration of the inhibitor. A higher STI concentration showed a longer lasting effect but lower effect in this study compared to that of TLCK. The protease inhibitors had some inhibitory effect on the synthesis and secretion of proteases, and interfered with the protease activity, thereby inhibiting the absorption of nutrients and delaying the growth and development of P. xylostella and reducing their ability to reproduce. These findings should provide the baseline information about using for effective pest management in the future.

Development of an immunochromatographic strip test for the rapid detection of soybean Bowman-Birk inhibitor

ABSTRACT Bowman-Birk inhibitor (BBI) is the main type of soybean trypsin inhibitor, which is a kind of antinutritional factor. It can cause indigestion and hinder growth and development of human and animal. Herein, a rapid immunochromatographic strip was developed in a sandwich format using the anti-BBI monoclonal antibody (mAb) in order to specifically detect BBI. The mAbs showed high affinity towards BBI and no cross-reactivity with other soybean allergenic proteins and antinutritional factors. The assay could be performed within 10 min. The limit of detection of the test strip was 0.5 µg/mL for visual and 0.23 µg/mL for strip reader. The assay showed high specificity for BBI and recovery efficacy of BBI in spiked milk samples ranged between 83.24% and 89.86% with the coefficient of variation of less than 7.98%. Therefore, the test strip could be used as a rapid and reliable detection method for BBI qualitative or quantitative assessment.

Structural insights into the unique inhibitory mechanism of Kunitz type trypsin inhibitor from Cicer arietinum L.

Kunitz-type trypsin inhibitors bind to the active pocket of trypsin causing its inhibition. Plant Kunitz-type inhibitors are thought to be important in defense, especially against insect pests. From sequence analysis of various Kunitz-type inhibitors from plants, we identified CaTI2 from chickpea as a unique variant lacking the functionally important arginine residue corresponding to the soybean trypsin inhibitor (STI) and having a distinct and unique inhibitory loop organization. To further explore the implications of these sequence variations, we obtained the crystal structure of recombinant CaTI2 at 2.8Å resolution. It is evident from the structure that the variations in the inhibitory loop facilitates non-substrate like binding of CaTI2 to trypsin, while the canonical inhibitor STI binds to trypsin in substrate like manner. Our results establish the unique mechanism of trypsin inhibition by CaTI2, which warrant further research into its substrate spectrum.AbbreviationsBApNANα-Benzoyl-L-arginine 4-nitroanilideBPTbovine pancreatic trypsinCaTI2Cicer arietinum L trypsin inhibitor 2DrTIDelonix regia Trypsin inhibitorEcTIEnterolobium contortisiliquum trypsin inhibitorETIErythrina caffra trypsin inhibitorKTIKunitz type inhibitorSTIsoybean trypsin inhibitorTKITamarindus indica Kunitz inhibitorCommunicated By Ramaswamy H. Sarma

Viscera of Labeo rohita: A Potential Source of Trypsin for Industrial Application

ABSTRACTThe serine protease trypsin was isolated and purified from the digestive system of carp Labeo rohita rohu by ammonium sulphate precipitation, ion exchange, and affinity chromatography. The purified enzyme showed high activity between pH 7.0 and 9.0. The activity was maximum at 40°C. Incubation of the purified enzyme with CaCl2 (2 mM) stabilized the enzyme activity for 8 h. The enzyme showed stability at 30 and 40°C for 1 h, but above 40°C, enzyme activity was reduced. The kinetic constants were recorded as Km (0.104 mM), kcat (44.25 s−1), and catalytic efficiency (427.54 s−1 mM−1). Monovalent, bivalent, and trivalent ions (Li+, K+, Hg2+, Al3+, Mg2+, Cd2+, Co2+, Zn2+, and Al3+) influenced the enzyme activity. Phenylmethylsulfonylflouride, soybean trypsin inhibitor, and N-α-p-tosyl-L-lysine chloromethyl ketone completely inhibited the enzyme activity, while ethylenediaminetetraacetate caused partial inhibition. Molecular mass of the purified enzyme was 22.46 kDa. The pH and temperature stability of enzyme may be useful for its industrial applications.

Extraction, partial purification and characterization of alkaline protease from rainbow trout (Oncorhynchus Mykiss) viscera

In this study, crude alkaline proteases were recovered from rainbow trout (Oncorhynchus mykiss) viscera and partially purified by use of different saturation of ammonium sulfate. The enzyme exhibited highest yield, purity and activity when precipitated at a saturation of 40–60% compared to other ranges of saturation. Molecular weight for extracted protease was between 8-24 kDa. The protease had caseinolytic activity over a wide range of temperatures (30-55 °C) and pH (4-12). Soybean trypsin inhibitor and trypsin-chymotrypsin inhibitor strongly inhibited the enzyme activity but it was stable in the presence of surfactant, oxidizing reagents and organic solvents. The proteases had serine protease activity but no collagenase activity was detected. The current study showed that partially purified protease from the digestive tract of rainbow trout could be applicable in food and detergent industry because of its good activity over a wide temperature and pH range and its good thermal stability.

Tranexamic acid blocks the thrombin-mediated delay of epidermal permeability barrier recovery induced by the cedar pollen allergen, Cry j1

We previously demonstrated that Cry j1, the major pollen allergen of Cryptomeria japonica (Japanese cedar), transiently increases protease activity and intracellular Ca2+ concentration in cultured human keratinocytes, and delays recovery after stratum corneum barrier disruption in human skin ex vivo. Topical application of tranexamic acid or trypsin-type serine protease inhibitors accelerates barrier recovery. We hypothesized that tranexamic acid might prevent the transient protease activity increase and the barrier recovery delay induced by Cry j1. Here, we tested this hypothesis and examined the mechanism involved. In cultured human keratinocytes, knock-down of protease-activated receptor 1 (PAR-1) reduced the transient increase of calcium induced by Cry j1, whereas knock-down of PAR-2 did not. Knock-down of thrombin significantly reduced the transient increases of calcium concentration and protease activity. Tranexamic acid, soybean trypsin inhibitor, or bivalirudin (a thrombin inhibitor) also reduced the calcium elevation induced by Cry j1 and/or thrombin. Co-application of tranexamic acid or bivalirudin with Cry j1 to human skin ex vivo blocked the delay of barrier recovery. These results suggest that thrombin and PAR-1 or PAR-1-like receptor might mediate the adverse effects of Cry j1 on human epidermal keratinocytes, and could open up a new strategy for treating inflammatory skin diseases.

The protease-based compensatory mechanism to minimize the effect of dietary Soybean Trypsin Inhibitor in Litopenaeus vannamei

Soybean meal is a recurrent ingredient in shrimp feed formulations; although economically convenient, the downside is the antinutritional molecules often present in its active forms. Recently, a midgut gland proteolytic activity adjustment to compensate the effect of increasing concentrations of soybean trypsin inhibitor (SBTI) delivered in shrimp feed was found in Litopenaeus vannamei; which prompted us to inquire on the transcriptional response of shrimp digestive peptidases against exogenous peptidase inhibitors and connect the results for a broader comprehension of the compensatory mechanism triggered by ingestion of SBTI. Our results indicate that such response involves at least two stages: in the first hours, a drastic increase in transcription of trypsin, an inhibitor sensitive protease was observed followed by a gradual increase in transcription of putatively less-sensitive protease chymotrypsin, and a non-sensitive metalloprotease was observed. At 23 h a second doses of SBTI was administered and at 24 h, trypsin transcripts returned to basal levels, whereas the transcription of genes encoding inhibitor-insensitive peptidases increased. Taken together, this paper is the first comprehensive study of the compensatory mechanisms to exogenous protease inhibitors in Penaeidae. Specific activity and gene expression of digestive proteases observe the same pattern, indicating a phenotypic plasticity in the digestive system of L. vannamei as an adaptive response to compensate the protein digestion capacity when some of the peptidase activities are reduced by the presence of protease inhibitors.

Extracellular protease trypsin activates amiloride-insensitive sodium channels in human leukemia cells.

Sodium influx is tightly regulated in the cells of blood origin. Amiloride-insensitive sodium channels were identified as one of the main sodium-transporting pathways in leukemia cells. To date, all known regulatory pathways of these channels are coupled with intracellular actin cytoskeleton dynamics. Here, to search for physiological mechanisms controlling epithelial Na+ channel (ENaC)-like channels, we utilized leukemia K562 cells as a unique model to examine single channel behavior in a whole-cell patch-clamp experiments. We have shown for the first time that extracellular serine protease trypsin directly activates sodium channels in plasma membrane of K562 cells. The whole-cell single current recordings clearly demonstrate no inhibition of trypsin-activated channels by amiloride or benzamil. Involvement of proteolytic cleavage in channel opening was confirmed in experiments with soybean trypsin inhibitor. More importantly, stabilization of F-actin with intracellular phalloidin did not prevent trypsin-induced channel activation indicating no implication of cytoskeleton rearrangements in stimulatory effect of extracellular protease. Our data reveals a novel mechanism modulating amiloride-insensitive ENaC-like channel activity and integral sodium permeability in leukemia cells.

Structural and functional analysis of miraculin-like protein from Vitis vinifera

The so-called miraculin-like proteins (MLPs) are homologous to miraculin, a homodimeric protein with taste-modifying activity that converts sourness into sweetness. The identity between MLPs and miraculin generally ranges from 30% to 55%, and both MLPs and miraculin are categorized into the Kunitz-type soybean trypsin inhibitor (STI) family. MLP from grape (Vitis vinifera; vvMLP) exhibits significant homology to miraculin (61% identity), suggesting that vvMLP possesses miraculin-like properties. The results of size-exclusion chromatography and sensory analysis illustrated that vvMLP exists as a monomer in solution with no detectable taste-modifying activity. Crystal structure determination revealed that vvMLP exists as a β-trefoil fold, similarly as other MLPs and Kunitz-type protein inhibitors. The conformation of the loops, including the so-called reactive loop in the STI family, was substantially different between vvMLP and STI. Recombinant vvMLP had inhibitory activity against trypsin (Ki = 13.7 μM), indicating that the protein can act as a moderate trypsin inhibitor.

Changes in soybean trypsin inhibitor by varying pressure and temperature of processing: A molecular modeling study

Abstract In recent years, molecular dynamic (MD) simulations have been used to understand the effects of various processing methods on the structural properties and stability of proteins. In this study, the conformational changes in soybean trypsin inhibitor molecule were evaluated with the use of high temperature and pressure. The MD simulations have been performed at various temperatures (300 K, 345 K and 373 K) and pressure (1 bar, 3 kbar, 6 kbar) combinations. The results showed that the soybean trypsin inhibitor (STI) molecule is relatively stable at high temperatures, primarily due to the presence of disulphide bonds. However, at higher pressures, significant compaction was observed. Root mean square deviations, Radius of gyration and volume of the STI molecule were evaluated. Furthermore, Ramachandran plots were used to evaluate the stability of the various simulated molecules. It was found that the compaction resulted in high steric interferences among the core residues.

Partial characterization of digestive proteases in adults of bigclaw river shrimp Macrobrachium carcinus

The present research was focused to characterize the digestive proteases in Macrobrachium carcinus adults using biochemical and electrophoretic techniques. Our results showed that the alkaline proteolytic activity from males and females did not show significant differences ( P > 0.05) between them, the optimum pH for digestive proteases is 8, and is very stable to changes in alkaline pH (8 and 10). The optimum temperature for alkaline proteases is 45oC and is stable from 25 to 45oC. The activity was totally inhibited with phenylmethylsulfonyl fluoride (PMSF), additionally the inhibition with trypsin soybean inhibitor 1 (SBT1) and tosyl-lysine-methyl ketone (TLCK) indicate high effect over serine proteases. Eight active bands were found using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) zymogram (range 17.8-94.0 kDa), which were partially inhibited with ovalbumin (Ovo), SBT1, phenanthroline (Phen), tosyl-phenylalanine-methyl ketone (TPCK), TLCK and ethylenediaminetetraacetic acid (EDTA), indicating an omnivorous digestive capacity, which remarks that the mainly alkaline protease in M. carcinus hepatopancreas are trypsin like enzymes.

Worm wounding increases levels of pollen-related food allergens in soybean (Glycine max)

The levels of food allergens in worm-wounded or non-wounded green soybeans (edamame) and mature soybeans were investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA), using allergen-specific antibodies. Non-wounded and worm-wounded soybeans showed similar total protein profiles after Coomassie brilliant blue staining, but some protein bands were observed to have been changed by worm wounding. Immunoblotting with specific antibodies for major soybean allergens (Gly m 5, Gly m 6, Gly m Bd 30K, and Kunitz soybean trypsin inhibitor) revealed that protein band profiles and intensities were not significantly changed by worm wounding. In contrast, levels of the pollen-related soybean allergens Gly m 4 and Gly m 3 were strongly increased by worm wounding in both green and mature soybeans, as detected by immunoblotting and ELISA. These results suggested that the pollen-related food allergen risk (i.e., oral allergy syndrome; OAS) from soybeans might be enhanced by worm wounding of soybeans.

Primary and passage cultures of astrocytes from rat brain cortex by using serum-free medium

Objective To explore the methods of primary and passage cultures of astrocytes by using serum-free medium. Methods Cells from cerebral cortices of Wistar rats within 24 h of birth were mechanically dispersed and cultured in serum-free medium. Reaching the confluence of 80%-90%, cells were digested by EDTA (ethylenediaminetetraacetic acid)-trypsin or trypsin, followed by 0.2 mg/ml or 1 mg/ml soybean trypsin inhibitor (SBTI) treatment and centrifugation for 3 min (3 000×g, 4 ℃) to stop digestion, then cells were subcultured with the ratio of 1 ∶2 or 1 ∶3. The cell phenotype was identified by immunofluorescence staining for glial fibrillary acidic protein (GFAP). The cell proliferation was detected by MTT assay. Results Under the phase-contrast microscopy, primary cells in serum-free medium were prosperous, showed high refractivity and demonstrated small cell body and nucleus with extensive processes. About 95% of the primary cells were positively stained for GFAP. The passage cells grew and proliferated well in the serum-free medium. The MTT results showed that the cell number in EDTA-trypsin group was higher than that in trypsin group (0.290±0.007 vs. 0.270±0.006, P<0.010). There was no difference in cell count either between 0.2 mg/ml or 1 mg/ml SBTI groups (0.290±0.013 vs. 0.290±0.017, P=0.820) or between 1 ∶2 and 1 ∶3 groups (0.340±0.070 vs. 0.320±0.030, P=0.770). Conclusion The results showed that we have established the method of primary and passage cultures of rat astrocytes by using serum-free medium, which may provide a new cell model for better evaluation of astrocyte function in vitro without interference of serum. Key words: Astrocytes; Rats; Culture media, serum-free; Primary cell culture; Passage culture

Effect of protease inhibitors on proteolytic degradation of rohu (Labeo rohita) gel

SummaryEndogenous proteases are responsible for gel weakening of fish muscle protein. We investigated inhibitory effect of four selected protease inhibitors: soya bean trypsin inhibitor, leupeptin, E64 and EDTA, to identify the protease groups responsible for gel degradation in both unwashed and washed rohu (Labeo rohita) gel. The protease inhibitory activity was determined by percentage inhibition and SDS‐PAGE pattern. Leupeptin and E64 showed a strong inhibition on the autolysis of unwashed gel, and myosin heavy chain (MHC) band was recovered by the addition of them. The results suggested that cysteine protease was the major endogenous protease involved in the autolysis. In the case of the washed gel, leupeptin and soya bean trypsin inhibitor were shown to have a strong inhibitory effect. Also, the addition of these inhibitors increased the intensity of the MHC band. The results implied that serine protease played an important role in the degradation of the gel network.

Culex pipiens sperm motility is initiated by a trypsin-like protease from male accessory glands.

In most animals, sperm are stored in a quiescent state in the male reproductive tract and only initiate motility when released into either the female reproductive tract, or, in the case of broadcast spawners, the external environment. Male accessory gland secretions transferred into the female reproductive tract may provide factors that modulate sperm viability and storage, or aid in sperm competition, as well as activate sperm motility. In several insects, serine proteases have been implicated in activating sperm motility. Our previous studies have shown that, in Culex quinquefasciatus, either a male accessory gland extract or purified trypsin is sufficient to initiate sperm motility in vitro. The objective of this study was to identify and characterize trypsin-like enzymes produced in the Culex male accessory glands. Mass spectrometry was used to analyze accessory gland proteins and this preliminary proteomic analysis identified 4 trypsin-like proteases (trypsin, trypsin4, and two trypsin7 isoforms). When measured with the chromogenic trypsin substrate Na -benzoyl-L-arginine-ethyl-ester-hydrochloride (BAEE), trypsin-like protease activity in the accessory glands was robust, with a pH optimum of 8. The pH range for the Culex trypsin activity was substantially narrower than a mammalian homologue (porcine pancreatic trypsin). A soybean trypsin inhibitor (SBTI) -agarose affinity column was used to independently identify trypsin-like accessory gland proteins. Several proteins were enriched in the eluate, as detected by silver staining of SDS-PAGE gels. Taken together, these data demonstrate the presence of trypsin-like activity and several trypsin-like proteins in the Culex male accessory glands.

Effects of goat milk fractions on the stability of IGF-I in simulated gastrointestinal conditions

The effects of goat milk fractions on the stability of insulin-like growth factor I (IGF-I) digested in the simulated gastrointestinal juice in vitro were investigated. IGF-I concentration was analyzed using an immunological sandwich-type ELISA method. The results showed that the concentration of pure IGF-I in simulated gastrointestinal juice decreased rapidly with the increasing digestion time. However, IGF-I in goat milk was more stable than pure IGF-I either in simulated gastric or intestinal juice. Goat milk and whey and whey supernatant fractions all effectively inhibited the proteolytic degradation of IGF-I in both the simulated gastric and intestinal juices. Dietary derived protease inhibitors, such as soya bean trypsin inhibitor (SBTI), lima bean trypsin inhibitor (LBTI) and egg white protease inhibitor (EWPI) all prevented IGF-I degradation in the simulated gastrointestinal juice, but the whey supernatant fraction represented more effective inhibition than the inhibitors. These results suggested that the whey supernatant fraction might contain specific inhibitors that protect IGF-I from the proteolytic degradation in the simulated gastrointestinal conditions, which was further verified by SDS-PAGE. Therefore, the whey supernatant fraction in goat milk could be a potential resource for the development of the effective oral IGF-I-based products.

Induction of proteases secreted by fusarium oxysporum F. SP. Dianthi in the presence of carnation root cell walls. Biochemical characterization of a serine protease

We determined that protease activity is secreted by Fusarium oxysporum f. sp. dianthi race 2 (Fod) in vitro. The cell wall extracted from resistant and susceptible cultivars of carnation (Dianthus caryophyllus L.) was used as an inducer. The activity increased in the presence of the two cultivars, and higher levels were detected with the susceptible cultivar. One of the three proteases secreted by Fod was purified using ion exchange (DEAE-cellulose), gel filtration (Sephadex G-75), and affinity chromatographies (Benzamidine Sepharose 6B). Using protease inhibitors, the purified enzyme was classified as a trypsin-like serine protease that was inhibited by phenylmethanesulfonyl fluoride, diisopropylfluorophosphate and the soybean trypsin inhibitor. In addition, the enzyme was biochemically characterized and determined to have an optimal pH of 8.0, an optimal temperature of 50°C, stability from pH 6 to 8, temperature stability between 40 to 60°C, a molecular weight of 54 kDa, and an isoelectric point of 4.4. The values of the Km and Vmax kinetic constants were found to be 0.31 mg/ml and 24.7 µmol/min, respectively.

An improved method of preparation of rat pancreatic acinar cells

Objective To explore a preferable method for preparation of rat pancreatic acinar cells. Methods Digestion solution consisting of collagenase and soybean trypsin inhibitor was injected into the pancreas parenchyma until well distended. The pancreas was cut into 2-4 mm pieces and incubated in digestion solution for 15 min. The incubation procedure was repeated 3 times. After filtration, centrifugation and isolation, pancreatic acinar cells containing 2-4 cell clusters were obtained. Results (7.3±0.5)×107 cells were obtained with the method above-mentioned from a rat. The cell viability was (95±3)% and cell purity was (97±1)%. Conclusion Here we provide a convenient, high-efficient way of isolating pancreatic acinar cells from rats with little effect on cell viability and function. Key words: Pancreas; Acinar cells; Isolation; Rat

Biochemical characterization a digestive trypsin in the midgut of large cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae).

A comprehensive study on digestive trypsin was undertaken in the larval midgut of Pieris brassicae L. Results of enzymatic compartmentalization showed a significantly higher activity of crude trypsin in the anterior larval midgut rather than posterior-midgut. Using Diethylaminoethyl cellulose fast flow column chromatography a purified trypsin was obtained by specific activity of 21 U mg-1 protein, recovery of 22%, purification fold of 28-fold and molecular weight of 25 kDa. This purified enzyme showed the highest activity at pH 8 and the corresponding temperature of 40°C. However, the specific inhibitors used including 4-(2-Aminoethyl) benzenesulfonyl fluroride hydrochloride, N-p-Tosyl-L-lysine methyl ester hydrochloride and Soybean Trypsin Inhibitor significantly lowered the activity of the purified enzyme in vitro. Moreover, the activity of trypsin and likewise the nutritional indices were significantly altered in the larval midgut feeding upon the leaves treated by 1 mM concentration of each inhibitor in comparison with control. Determination of enzymatic characteristics of insect trypsins is crucial in paving the path for controlling pests by potential natural compounds via transgenic plants.