Kunitz Soybean Trypsin Inhibitor Research Articles

Detection of Two New Variants of Soybean Kunitz Trypsin Inhibitor through Electrophoresis.

In order to detect mobility variants of soybean Kunitz trypsin inhibitor (SKTI) proteins, we developed a large slab gel method (18.5 cm long X 14.5 cm wide) of elec-trophoresis, and analyzed 173 cultivars of soybeans (Glycine max ) from Japan and China, and 62 populations of wild soybeans (G. soja ) collected from all over lwate Prefecture, Japan. As a result of this electrophoretic survey, two new mobility variants were discovered: a variant showing a slightly slower mobility than the Tia type (tentatively designated as Tia-s ) in a wild soybean line and another variant with a slightly faster mobility than the Tib type (tentatively designated as Tib-f) in a cultivated soybean variety. Transmissibility of this variation to consecutive generations was confirmed by parent-offspring tests and hybridization tests using the variants and/or the common types. Isoelectric focusing (IEF) test applied to these two variants revealed differences in the location of the isoelet-ric points in the gel. Significance of these discoveries was discussed.

Characterization of Proteintype Proteinase Inhibitors by High Performance Capillary Electrophoresis

Abstract A method based on high performance capillary electrophoresis (HPCE) has been developed for identification and quantitative determination of inhibitors from leguminous seeds. The method allows specific characterization of Kunitz soybean trypsin inhibitor (KSTI) and Bowman Birk inhibitor (BBI) from soybean (Glycine max., L.). Standard curves for the inhibitors showed a satisfactory linearity between normalized peak area and sample concentration, and a good repeatability of the method was found. Identification of protein peaks as proteinase inhibitors was based on their binding to trypsin. The system also proved useful for determination of the number and specificity of inhibitor sites on the individual inhibitors and for binding studies with monoclonal antibodies against the inhibitors.

Dietary proteinase inhibitors alter complement of midgut proteases

Plant serine proteinase inhibitors (Pls) have the potential to restrict the growth and/or development of herbivorous insects. However, there are limitations to the efficacy of these Pls. An insect's susceptibility to a Pl is determined, at least in part, by the relative proportion of proteolytic enzyme activity in the midgut that is suppressed by that inhibitor. Insects adapt to dietary trypsin inhibitor in their host plant by secreting “inhibitor-resistant” trypsin(s). These “inhibitor-resistant” enzyme(s) may be the standard proteinase(s) secreted into the midgut (e.g., Pieris rapae), or may be enhanced following ingestion of proteinase inhibitor (e.g., Helicoverpa zea). In addition, insects may be pre-adapted to specific Pl(s), following adaptation to a Pl from the same family. For example, Pieris rapae is a crucifer specialist that is resistant to cabbage Pl, but is also resistant to Kunitz soybean trypsin inhibitor, a Pl in the same family as cabbage Pl, but from a non-host plant. The ultimate value of this pre-adaptation to herbivory by a species of insect will be determined by the number of different families of Pl in host plant(s) to which the species has adapted, and the distribution of those families among other species of plants. Thus, it is possible that the presence of a plant Pl limits herbivory by insect(s). However, multiple inhibitors, matched to the complement of enzymes in the insect's midgut, may be required to enhance this resistance of plants to herbivorous insects. © 1996 Wiley-Liss, Inc.

Dietary proteinase inhibitors alter complement of midgut proteases

Plant serine proteinase inhibitors (Pls) have the potential to restrict the growth and/or development of herbivorous insects. However, there are limitations to the efficacy of these Pls. An insect's susceptibility to a Pl is determined, at least in part, by the relative proportion of proteolytic enzyme activity in the midgut that is suppressed by that inhibitor. Insects adapt to dietary trypsin inhibitor in their host plant by secreting “inhibitor-resistant” trypsin(s). These “inhibitor-resistant” enzyme(s) may be the standard proteinase(s) secreted into the midgut (e.g., Pieris rapae), or may be enhanced following ingestion of proteinase inhibitor (e.g., Helicoverpa zea). In addition, insects may be pre-adapted to specific Pl(s), following adaptation to a Pl from the same family. For example, Pieris rapae is a crucifer specialist that is resistant to cabbage Pl, but is also resistant to Kunitz soybean trypsin inhibitor, a Pl in the same family as cabbage Pl, but from a non-host plant. The ultimate value of this pre-adaptation to herbivory by a species of insect will be determined by the number of different families of Pl in host plant(s) to which the species has adapted, and the distribution of those families among other species of plants. Thus, it is possible that the presence of a plant Pl limits herbivory by insect(s). However, multiple inhibitors, matched to the complement of enzymes in the insect's midgut, may be required to enhance this resistance of plants to herbivorous insects. © 1996 Wiley-Liss, Inc.

Optimization of hapten-protein conjugation by high-performance capillary electrophoresis

HPCE has been found to be efficient for the optimization of conjugation procedures employed for coupling of the haptens p- nitrophenyl-α- d - galactopyranoside (PNPG) and soyasaponin I to a carrier protein, Kunitz soybean trypsin inhibitor (KSTI). The carbohydrate moieties of the haptens were oxidized with periodate followed by reaction with ϵ-amino groups of the carrier. For PNPG, the periodate oxidations (0.01–0.2 M NaIO 4) were followed by HPCE and 0.1 M periodate was chosen as the optimum concentration. The coupling of PNPG to KSTI was found to proceed at a constant rate for more than 250 min. The reaction rate for the soyasaponin conjugation to KSTI declined after 80 min of incubation. The coupling of soyasaponin to KSTI was confirmed by enzyme-linked immunosorbent assay with monoclonal antibodies directed against soyasaponin I. The method was found to be particularly powerful for the investigation of conjugates with low epitope densities that are difficult to determine otherwise.

Effects of the soybean (Kunitz) trypsin inhibitor on growth and digestive proteases of larvae of Spodoptera litura

Extracts from the digestive tract of final instar larvae of Spodoptera litura, reared on artificial diet, contained trypsin-like (BApNA-hydrolysing) activity with a pH optimum of 10.5, an elastase-like (SA 2PLpNA-hydrolysing) activity with a pH optimum of 10.5 and leucine aminopeptidase activity with a pH optimum of 8.5. No chymotrypsin amidase-like (BTpNA-hydrolysing) activity could be detected, but chymotrypsin esterase-like (BTEE-hydrolysing) activity was measured with a pH optimum of 8.5. Titration, in vitro of the soybean (Kunitz) trypsin inhibitor (SBTI) against each enzyme at its optimal pH revealed that the inhibitor was most effective at retarding the trypsin-like activity, only slightly effective against the elastase-like and chymotrypsin esterase-like activity, but completely ineffective against leucine aminopeptidase. Incorporation of SBTI into the diet of neonate larvae at 0.2% (w/v) and 0.5% (w/v) retarded growth rate when compared with larvae fed artifical diet only. The observed differences in growth rate occurred within the first three days, resulted in weight differences that were sustained over the whole period of the trial (12 days) and correlated with inhibitor concentration; the slowest growth rate and the lower weights were observed for larvae fed with the higher SBTI concentration (0.5%) in their diet. Assay of the digestive proteases in these larvae demonstrated that only the trypsin-like activity was stimulated significantly in larvae fed on diet containing SBTI.

Inactivation of proteinaceous protease inhibitors of soybeans by isolated fungi

Proteinaceous protease inhibitors, Kunitz Soybean Trypsin Inhibitor (KSTI) and Bowman Birk Inhibitor (BBI), in legume seeds reduce the digestibility of proteins in feed of monogastric animals. Enzymatic inactivation of these inhibitors will increase the nutritional value of the feed. The aim of this study was to obtain microorganisms which produce proteases which specifically inactivate the inhibitors under conditions that occur in the stomach. Hereto, microorganisms were enriched at pH 3.0 and 37 °C on KSTI or BBI as sole nitrogen and sulphur source. Nine fungal strains were isolated from the enrichment cultures. The isolated strains were able to inactivate both inhibitors. KSTI was inactivated more effectively than BBI. KSTI inactivation was pH dependent. At pH 5.5, only 30–45% of the KSTI was inactivated in 4 days, whereas, 80–90% of the KSTI was inactivated in 2 days at pH 3.0. The isolated strains varied in capacities to inactivate KSTI. In general, strains which inactivated KSTI to a higher extent had higher biomass yields and higher general protease activities. The KSTI specificity of the most promising strain (K1) which had a relatively high KSTI inactivation capacity and a relatively low general protease activity at pH 3.0, was studied in more detail. The KSTI specificity of the produced proteases was affected by pH and by the type of substrates used [KSTI, bovine serum albumin fraction V (BSA), cysteine or without substrate]. KSTI specificity of the proteases produced at pH 5.5 was low as compared to pH 3.0. The highest KSTI specificity, 1.7 mole KSTI per mole albumin, was found when strain K1 was grown on cysteine at pH 3.0.

Characterization of Monoclonal Antibodies that Recognize the Soybean (Kunitz) Trypsin Inhibitor: Binding to the Inhibitor Interrupts the Formation of the Trypsin : Inhibitor Complex

Summary Three monoclonal antibodies (mAbs) have been raised against the soybean (Kunitz) trypsin inhibitor (SBTI), and designated 5H-10, 6D-12, and 7B-11 . Isotyping revealed that all three are IgG variants. Western blotting analysis has shown that (i) each monoclonal antibody specifically recognizes SBTI, but not the Bowman-Birk inhibitor (BBI) from soybean, and (ii) mAbs 5H-10 and 6D-12 recognize a peptide of the correct molecular weight in seeds of two soybean cultivars that contain distinct allelic forms of the inhibitor (designated as Ti a and Ti b ). However, binding studies using ELISA have shown that the affinity for purified SBTI differs between the clones. Further, mAb 6D-12 can interrupt the inhibition of trypsin by SBTI such that the activity of the proteinase is restored, while the other two monoclonals have no apparent effect. These results suggest that the epitope for mAb 6D-12 differs from mAbs 5H-10 and 7B-11, and as such this set o f monoclonal antibodies can be of use in further characterising the trypsin : SBTI interaction.

Proteolysis of Kunitz Soybean Trypsin Inhibitor. Influence on Its Activity

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProteolysis of Kunitz Soybean Trypsin Inhibitor. Influence on Its ActivityIosif A. Vaintraub and Haram B. YattaraCite this: J. Agric. Food Chem. 1995, 43, 4, 862–866Publication Date (Print):April 1, 1995Publication History Published online1 May 2002Published inissue 1 April 1995https://doi.org/10.1021/jf00052a003RIGHTS & PERMISSIONSArticle Views111Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Isolation of a new member of the soybean Kunitz-type proteinase inhibitors.

Proteinase inhibitors are present in almost a11 organisms (Ryan, 1981). Most of these proteins are specific in their interaction with proteinases, inhibiting the proteolytic activity. In plants, this protein family is highly abundant in seeds and tubers of many species. Since high levels of proteinase inhibitor in agricultura1 crops may result in alterations of the digestive enzymes in human and animals, research has been targeted toward modification of the proteinase inhibitor levels in those plant species (Ryan, 1981). In soybean (Glycine max), among others, two important families of proteinase inhibitors have been characterized: the Kunitz inhibitor, which shows specificity for trypsin (Ryan, 1981; Kim et al., 1985), and the Bowman-Risk, which inhibits trypsin, chymotrypsin, and elastase (Ryan, 1981, 1988; Tan-Wilson, 1988). These proteins serve an important function in storage protein metabolism by regulating the level of proteinase activity during seed development (Ryan, 1988). It has been shown that they also play an important role against pathogen attack by acting as endogenous insecticides (Hilder et al., 1986). During screening of a Agtll cDNA library, we isolated a cDNA clone with an insert size of 802 bp. This insert contains a unique open reading frame of 624 bp, which encoded for a 208-amino acid polypeptide (Table I). Sequence comparison with other proteins contained in the data base shows a high level of homology to the Kunitz trypsin inhibitor family. Southern blot analysis of the soybean Kunitz trypsin inhibitor indicates that this protein family contains at least 10 members, some of which are organized in tandem pairs (Jofuku and Goldberg, 1989). At present, four of these members have been cloned and characterized (Jofuku and Goldberg, 1989; Song et al., 1993). The KTi3 and KTi-b genes encode for the major Kunitz trypsin inhibitor proteins, Tia and Tib, respectively (Kim et al., 1985). Studies of the active site indicated that two amino acids, (Pl) and Ile64 (Pl’), are essential for activity. Thus, trypsin inhibitor activity is detected only Table 1. Characteristics of a cDNA coding for a Kun ih trypsin inhibitor orotein. KTi-S

Duodenase, a New Serine Protease of Unusual Specificity from Bovine Duodenal Mucosa. Purification and Properties

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl-cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 +/- 0.5 kDa by SDS/PAGE and G-2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 +/- 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars. The enzyme displays trypsin-like and chymotrypsin-like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9-8.2. Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an active-site serine in this protease. alpha-N-Tosyl-L-lysine chloromethane and alpha-N-tosyl-L-phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin-like and chymotrypsin-like activities of duodenase. The enzyme activity was strongly suppressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity. The N-terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T-lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases. The biological role of duodenase is discussed.

High-performance capillary electrophoresis for characterization of hapten—protein conjugates used for production of antibodies against soyasaponin I

Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten—protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea ( Pisum sativum L.) and soybean [ Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten ( p-nitrophenyl-α- d-galactoside; PNPG) coupled to KSTI. The PNPG—KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten—protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.

High-performance capillary electrophoresis for the determination of trypsin and chymotrypsin inhibitors and their association with trypsin, chymotrypsi

High-performance capillary electrophoresis (HPCE) was adapted for the determination of Kunitz soybean trypsin inhibitor, Bowman Birk inhibitor from soybean and protein-type proteinase inhibitors from pea ( Pisum sativum L.). The method was developed for the determination and characterization of the inhibitors, the enzymes trypsin and chymotrypsin and the monoclonal antibodies (mAbs) raised against the inhibitors, and also the inhibitor—enzyme and inhibitor—mAb association complexes. The results from studies involving the use of various types of buffers revealed the advantages of having zwitterions such as trimethylammoniumpropyl sulphonate (AccuPure) or taurine included in the buffer. The use of capillaries dynamically coated with zwitterions efficiently reduced the interactions of the proteins with the silica capillary surface, which was important for the analyses for trypsin, chymotrypsin and mAbs and their association complexes with the inhibitors. The influence of temperature, voltage, pH and buffer type on migration times, resolution, peak areas and number of theoretical plates was investigated for the proteins studied. The proposed HPCE method is very suitable for studies of proteinase inhibitors compared with traditional inhibitor studies, and it gives efficient protein separations with the possibility of 245 000 plates/m.

Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera

Soybean Kunitz trypsin inhibitor (SBTI) and soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) were incorporated into artificial diet and fed to newly moulted 3rd-instar larvae of Helicoverpa armigera, the corn earworm. 1. 1. Neither SBTI nor SBBI affected larval survival for 14 days. After this time, many of the larvae fed diets with high levels of inhibitor suddenly died. This effect was most marked with SBTI diets. 2. 2. When compared with the effect of the control diet (no inhibitor), inhibitors in the diet reduced total larval biomass and mean larval weight. This effect on larval growth was much greater with dietary SBTI than with SBBI. The relative effectiveness of the inhibitors in vitro is in agreement with previous data showing that SBTI was a more effective inhibitor of the H. armigera trypsin-like gut protease in vitro. 3. 3. The presence of inhibitor in the diet reduced the mean larval weight at moult, suggesting a possible response of ecdysis to semi-starvation. This effect was greater with SBTI than with SBBI. 4. 4. Faecal output was used as a measure of food intake. By day 10 of the feeding trials, the mean faecal output/larva was similar for larvae fed the control and SBBI diets, but it was significantly lower for larvae fed dietary SBTI. However, when faecal output was corrected to account for developmental states and weights of larvae, no differences were found between the effects of control and inhibitor diets. 5. 5. Feeding larvae a diet containing 0.234 mM SBTI significantly reduced the trypsin-like enzyme activities found in their gut contents when compared with the levels found in larvae fed the control diet. The possible mechanisms of action of SBTI and SBBI in vivo are discussed, as are their relative potentials for protecting crop plants against damage by larval H. armigera.

Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum

1. Combinations of a cysteine proteinase inhibitor (CPI) and serine proteinase inhibitors (SPI) in wheat germ diets were toxic to larvae of the red flour beetle, Tribolium castaneum, when tested at levels where individual inhibitors were nontoxic. 2. Mixtures of 0.1% (w/w) CPI (E-64) plus 1% of either of three plant SPIs (soybean Kunitz trypsin inhibitor, soybean Bowman-Birk trypsin-chymotrypsin inhibitor, or lima bean trypsin inhibitor) inhibited T. castaneum growth, resulting in 82–97% reduction in larval weight gain 17 days after hatching and 40–60% mortality. 3. Supplemention of diet containing 0.1% E-64 plus 1% soybean Kunitz trypsin inhibitor (STI) with a mixture of amino acids at 7% caused a partial reversal of the growth inhibition, with 91% of the larvae surviving. 4. Diet containing 0.1% E-64 plus either 5 or 10% STI resulted in 100% mortality of the larvae during the first or second instar. 5. Addition of a mixture ofamino acids at 20% to the 0.1% E-64 plus 10% STI diet allowed 89% of the larvae to develop into adults. 6. The synergism between different classes of proteinase inhibitors in the insect's diet that enhances growth inhibition and toxicity demonstrates the potential for an insect pest management strategy involving the coordinated manipulation of two or more types of digestive enzyme inhibitor genes in plants.

Construction of Insect Resistant Transgenic Tobacco Plants by Expression of Soybean Kunitz Trypsin Inhibitor

Construction of Insect Resistant Transgenic Tobacco Plants by Expression of Soybean Kunitz Trypsin Inhibitor

Proteases catalysing processing and degradation of kunitz soybean trypsin inhibitor during seed maturation

Examination of the levels of the Kunitz soybean trypsin inhibitor (KSTI) in the seed at various stages of seed maturation shows a slow phase of accumulation followed by a rapid phase when the seeds have reached half their maximum fresh weight and 83% of maximum length. The rapid accumulation is coincident with two changes in the activities of proteases relating to the Kunitz soybean trypsin inhibitor. One change is a decline in the activity of a protease catalysing the conversion of the KSTI to a form with an M r 5 500 smaller than the mature form found in the dry seed. This form eventually disappears, suggesting it to be an intermediate in a proteolytic degradation of the Kunitz inhibitor. The second change is an increase in the activity of a protease catalysing the proteolytic processing of the precursor form to the mature form of the KSTI. Both the precursor form of KSTI and the proteolytic degradation intermediate are found in vivo, indicating that the enzyme activities found in seed extracts are indeed functioning in the intact developing seed.

Trypsin Inhibitor from the Seeds of Acacia confusa1

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.

Initiation of the Degradation of the Soybean Kunitz and Bowman-Birk Trypsin Inhibitors by a Cysteine Protease

Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti(a), to Ti(a) (m), the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the alpha, alpha', and beta subunits of soybean beta-conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon.

Characterization of a Human Trypsin Resistant to Kunitz Soybean Trypsin Inhibitor: Studies of Duodenal Juices after Tube Instillation of Raw Soybean Extract

Human duodenal juices collected during tube instillation of raw soybean extract into the duodenum contained free trypsin and free Kuntiz soybean trypsin inhibitor (KTI) in the simultaneous presence of trypsin-KTI complexes. It has previously been suggested that this KTI-non-inhibitable trypsin has a general resistance to serine protease inhibitors. Four different trypsin forms have been found and partly characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing followed by Western immunoblotting or enzyme staining. In addition, crossed immunoelectrophoresis and affinity chromatography with antibody-coupled gels have been used for identification of free and inhibitor-complexed trypsin.