Kunitz Soybean Trypsin Inhibitor Research Articles

4959310 Monoclonal antibodies to soybean Kunitz trypsin inhibitor and immunoassay methods: David Brandon, Anne H Bates, Mende Friedman assigned to The United States of America as represented by the Secretary of Agriculture

4959310 Monoclonal antibodies to soybean Kunitz trypsin inhibitor and immunoassay methods: David Brandon, Anne H Bates, Mende Friedman assigned to The United States of America as represented by the Secretary of Agriculture

4959310 Monoclonal antibodies to soybean kunitz trypsin inhibitor and immunoassay methods: David Brandon, Anne H Bates, Mende Friedman assigned to The United States of America as represented by the Secretary of Agriculture

4959310 Monoclonal antibodies to soybean kunitz trypsin inhibitor and immunoassay methods: David Brandon, Anne H Bates, Mende Friedman assigned to The United States of America as represented by the Secretary of Agriculture

Amino acid sequence of the acidic kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC

The primary sequence of trypsin inhibitor-2 (WBTI-2) from Psophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 from Erythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors from Adenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that the Psophocarpus and Erythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.

Effect of Maillard browning reactions of the Kunitz soybean trypsin inhibitor on its interaction with monoclonal antibodies

Carbohydrates react with proteins to form nonenzymatically browned products, including Maillard reaction products. The effects of these transformations on the antigenicity of the Kunitz soybean trypsin inhibitor (KTI) were studied with use of two monoclonal antibodies. Solid mixtures of KTI and carbohydrates were heated in an oven at 120 degrees C, dialyzed, freeze-dried, and analyzed by enzyme-linked immunosorbent assay (ELISA). Glucose, lactose, and maltose decreased the antigenicity of KTI by 60-80%, compared to a control sample heated without carbohydrate. Starch was less effective than the three reducing sugars. This decrease was rapid, occurring within 10 min when glucose was heated with KTI, with retention of 60% of the chemically available lysine. Longer heating times increased browning and reduced the level of available lysine in KTI, without further reducing antigenicity. The results suggest that relatively mild conditions of heating food proteins with carbohydrates can reduce the antigenicity of the protein and possibly modify sites known to elicit allergenic responses.

Kunitz trypsin inhibitor genes are differentially expressed during the soybean life cycle and in transformed tobacco plants.

We investigated the structure, organization, and developmental regulation of soybean Kunitz trypsin inhibitor genes. The Kunitz trypsin inhibitor gene family contains at least 10 members, many of which are closely linked in tandem pairs. Three Kunitz trypsin inhibitor genes, designated as KTi1, KTi2, and KTi3, do not contain intervening sequences, and are expressed during embryogenesis and in the mature plant. The KTi1 and KTi2 genes have nearly identical nucleotide sequences, are expressed at different levels during embryogenesis, are represented in leaf, root, and stem mRNAs, and probably do not encode proteins with trypsin inhibitor activity. By contrast, the KTi3 gene has diverged 20% from the KTi1 and KTi2 genes, and encodes the prominent Kunitz trypsin inhibitor found in soybean seeds. The KTi3 gene has the highest expression level during embryogenesis, and is also represented in leaf mRNA. All three Kunitz trypsin inhibitor genes are regulated correctly in transformed tobacco plants. Our results suggest that Kunitz trypsin inhibitor genes contain different combinations of cis-control elements that program distinct qualitative and quantitative expression patterns during the soybean life cycle.

Hypertrophy and hyperplasia of the rat pancreas produced by short-term dietary administration of soya-derived protein and soybean trypsin inhibitor.

Feeding soy protein concentrate to weanling rats over a one-week period produced a dose-related increase in pancreatic weight due to an increase in acinar cell size. Hyperplastic changes occur simultaneously, as evidenced by an increase in mitotic activity after two days on the test diet. Similar changes were also obtained by feeding soybean Kunitz trypsin inhibitor over the same time period. The results suggest that this approach may be useful as a model to investigate the effect of plant-derived material on the pancreas, in particular proliferative lesions.

A frameshift mutation prevents Kunitz trypsin inhibitor mRNA accumulation in soybean embryos.

We investigated the molecular basis of a soybean Kunitz trypsin inhibitor (KTi) gene mutation that prevents the accumulation of Kunitz trypsin inhibitor protein during seed development. We found that mRNA encoding the major Kunitz trypsin inhibitor protein (KTi3 mRNA) is reduced at least 100-fold in null (KTi-) embryos but that KTi3 gene transcriptional activity is similar in Kunitz trypsin inhibitor producing embryos (KTi+) and in KTi- embryos. We sequenced the Kunitz trypsin inhibitor KTi3 gene from both KTi3+ and KTi3- lines and found that these genes differ by only three nucleotides (+481, +486, and +487) within the KTi3 coding region. Alteration of these nucleotides results in a frameshift within the KTi3- gene that causes premature termination during translation. Our results suggest that the KTi3- frameshift mutation results in KTi3- mRNA destabilization and leads to a drastic reduction in KTi3 mRNA prevalence.

Definition of functional and antibody-binding sites on Kunitz soybean trypsin inhibitor isoforms using monoclonal antibodies

Elaboration d'un test ELISA pour la caracterisation et l'etude de la structure de ces inhibiteurs d'enzyme

Survey of the Proteolytic Activities Degrading the Kunitz Trypsin Inhibitor and Glycinin in Germinating Soybeans (Glycine max)

The cotyledons of the soybean (Glycine max [L.] Merrill cv Amsoy 71) were examined for proteolytic activities capable of degrading soybean seed proteins. Three distinct activities were identified that attack the native Kunitz soybean trypsin inhibitor of Amsoy 71, Ti(a). Protease K1 cleaves Ti(a) to Ti(a) (m), the inhibitor form lacking the five carboxyl-terminal amino acid residues relative to Ti(a). Protease K1 is a cysteine protease that peaks in activity on day 4 after the beginning of imbibition, with maximal activity toward Ti(a) at pH 4. The characteristics of protease K1 are consistent with the involvement of this protease in the initial proteolysis of the Kunitz inhibitor during germination. Protease K2 also degrades Ti(a) at pH 4 but produces no electrophoretically recognizable products. It peaks later in seedling growth, at day 8. Protease K3 degrades Ti(a) to products other than Ti(a) (m). However, it is active at pH 8. Two proteolytic activities were identified that attack the major storage protein, glycinin. Protease G1 (which appears by 4 days after imbibition) specifically cleaves the acidic polypeptides of glycinin at pH 4, yielding a product approximately 1.5 kilodaltons smaller. Protease G1 is inhibited by metal chelators as well as by reagents reactive toward thiols. Protease G2 also degrades the glycinin acidic chains at pH 4, but without the appearance of electrophoretically recognizable products. Protease G2, while present at low levels in the dry seed, is found primarily in the cotyledons after 8 days of growth.

Intramolecular disulfide loop formation in a peptide containing two cysteines.

The cyanogen bromide fragment comprising residues 115-181 of Kunitz soybean trypsin inhibitor is a soluble random-coil peptide at pH 7 containing two cysteines separated by eight other amino acids in the primary sequence. Four of the six rate constants have been determined for the three disulfide exchange reactions between this fragment and oxidized and reduced forms of N-acetylcysteine methyl ester. The rate constant for intramolecular loop formation in the fragment containing one thiolate anion and one sulfur connected by a disulfide bond to the small cysteine analogue is 0.36 +/- 0.15 s-1 at 23 degrees C in 3 M guanidine hydrochloride. This measurement provides a frame of reference corresponding to formation of a small but sterically unstrained loop, the fast limit for intramolecular disulfide exchange in a random-coil peptide.

Chymotrypsin-specific protease inhibitors decrease H2O2 formation by activated human polymorphonuclear leukocytes.

Stimulated phagocytic cells generate active oxygen species which are known to contribute to inflammatory diseases, necrosis of surrounding tissues, mutagenicity and carcinogenicity. Until now, it was not certain whether protease inhibitors are capable of decreasing the production of those oxygen species, and if they are, what type of protease inhibitor is the most active. In this work we monitored formation of H2O2 by 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated polymorphonuclear leukocytes (PMNs) because H2O2 is the immediate precursor of the actual damaging species. These determinations were carried out in the absence or presence of protease inhibitors and/or superoxide dismutase (SOD). The protease inhibitors tested were: potato inhibitors 1 (PtI-1) and 2 (PtI-2), a chymotrypsin-inhibitory fragment of PtI-2 (PCI-2), chicken ovoinhibitor (COI), turkey ovomucoid ovoinhibitor (TOOI), Bowman-Birk inhibitor (BBI), lima bean inhibitor (LBI) and soybean (Kunitz) trypsin inhibitor (SBTI). The order of activity, as measured by inhibition of H2O2 formation by TPA-activated PMNs during incubation at 37 degrees C for 30 min, was (in descending order): PtI-1 greater than or equal to PCI-2 greater than PtI-2 greater than COI greater than BBI greater than or equal to TOOI greater than LBI greater than SBTI. Thus, the most effective were the chymotrypsin-specific inhibitors PtI-1 and PCI-2, followed by the bifunctional inhibitors recognizing both chymotrypsin and trypsin, and the least active was SBTI, a predominantly trypsin inhibitor. At the higher concentrations of protease inhibitors tested, the inhibitory activity was similar in both the absence and presence of SOD. These results show that protease inhibitors specific for chymotrypsin but not those that are trypsin-specific are capable of inhibiting formation of active oxygen species during the oxidative burst of stimulated human PMNs.

Photoreactive, active derivatives of trypsin and chymotrypsin inhibitors from soybeans and chickpeas.

The photoreactive arylsufenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (STI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-STI indicated that only one of the two tryptophans, 93 or 117, present in STI was modified. Amino acid analysis of the two separated CNBr-cleavage products of 2,4-NAPS-STI showed that only tryptophan 93 underwent modification. 2,4-NAPS-STI fully retained its inhibitory activity against trypsin. The covalent attachment of 2,4-NAPS-STI to tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride. Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas were prepared by selective modification of the epsilon-amino groups of 2,4(5)-NAPS-Cl. The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.

Proteolysis of kunitz soybean trypsin inhibitor during germination

During the germination and seedling growth of soybeans ( Glycine max) a new form of the Kunitz soybean trypsin inhibitor (KSTI) appears in the cotyledons distinct from the KSTI of the quiescent seed. This inhibitor has been purified from the germinated seeds of soybean cultivar Amsoy 71 and cultivar Fiskeby V. These cultivars contain the Ti a and Ti i variants of KSTI respectively in the dry seed. The inhibitors were characterized by sodium dodecylsulphate-polyacrylamide gel electrophoresis, amino acid analysis, and partial sequence determination. The newly appearing form in Amsoy 71 (Ti a m) was found to be identical to Ti a except for the loss of the five residue sequence at the carboxylterminus of Ti a. The Ti b variant of KSTI was found to contain at least 199 amino acid residues (as opposed to 181 for Ti a). The amino-terminal sequence of Ti b was found to be identical to that of Ti a for the first ten residues. The KSTI species appearing in Fiskeby V with germination, Ti b m, appears to be derived from Ti b by the loss of the carboxylterminal decapeptide.

Differential Expression of Kunitz and Bowman-Birk Soybean Proteinase Inhibitors in Plant and Callus Tissue

Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred.

Purification and some properties of rabbit liver cytosol thioltransferase.

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.

Purification and some properties of bovine liver cytosol thioltransferase.

A cytosol thioltransferase was purified 37,000-fold from bovine liver by essentially the same procedure as reported for rat liver enzyme by Axelsson et al. [1978) Biochemistry 17, 2978-2984). The purified enzyme appears to be homogeneous on sodium dodecyl sulfate (SDS)-gel electrophoresis and has a molecular weight (Mr) of 11,000, an isoelectric point (pI) of 8.1, and an optimum pH with S-sulfocysteine and GSH as substrates of 8.5. It is specific for disulfides including L-cystine, S-sulfocysteine, ribonuclease A, trypsin, soybean kunitz trypsin inhibitor, soybean Bowman Birk trypsin inhibitor and insulin, and converts Bowman Birk trypsin inhibitor to an inactive form. The enzyme does not act as a protein : disulfide isomerase, as measured by reactivation of "scramble" ribonuclease and Kunitz soybean trypsin inhibitor. Thioltransferase activity was found in cytosol of various bovine tissues.

Photoreactive derivative of Kunitz's soybean trypsin inhibitor.

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1-114 and 115-181. Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [3H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.

Structure of soybean Kunitz trypsin inhibitor mRNA determined from cDNA by using oligodeoxynucleotide primers

Oligodeoxynucleotides complementary to the deduced mRNA sequence of soybean Kunitz trypsin inhibitor (KTI) were used to prime the synthesis of cDNA from soybean cotyledon total poly(A) RNA. The primed cDNA was used to select clones from a Glycine max cotyledon cDNA library. Two out of twelve hybridizing clones were shown to contain KTI cDNA. The nucleotide sequence of one clone, pSTI 9-2, was determined and it was found to encompass the complete protein coding region of KTI excet for three C-terminal residues. Trypsin inhibitor is synthesized with a 25 amino acid hydrophobic N-terminal sequence presumed to be a signal peptide. The mature polypeptide encoded by pSTI 9-2 agrees with the published amino acid composition of KTI, but contains two discrepancies at the peptide sequence level.

Use of local electrostatic environments of cysteines to enhance formation of a desired species in a reversible disulfide exchange reaction

The role of electrostatic factors has been evaluated for the reversible disulfide exchange reaction between N-acetylcysteine (A) and a peptide fragment (B) comprising residues 85–114 of Kunitz soybean trypsin inhibitor. In A, the sulfhydryl group has a negative carboxyl neighbor on the cysteine itself. In B, the only charged group within five residues of the single cysteine at position 86 is the positive N-terminal amino group on residue 85. The concentrations of the monomers A and B and of the disulfides AA, AB and BB have been determined as a function of time in kinetic experiments at pH 7, 23°C and ionic strengths of 20 mM and 1 M. At both ionic strengths the sulfhydryl acid dissociation constants K a have been determined for A and B, as well as the four rate constants for the disulfide exchange reaction. The electrostatic effects are small in magnitude but occur in expected directions. Local cysteine environments enhance formation of the mixed disulfide (AB), having a favorable configuration of adjacent unlike charges and generate decreases in the AA and BB disulfides joining regions of identical charge. These experiments represent an initial step towards use of intrinsic protein functional groups to direct formation of specific disulfides in a synthetic protein.

Inhibition of superoxide production in human neutrophils by purified soybean polypeptides. Re-evaluation of the involvement of proteases.

Inhibition of neutrophil superoxide production has been previously reported for reagents and polypeptides which also inhibit serine proteases. There are disagreements between the results of different laboratories including our own, which have attempted to use the Kunitz soybean trypsin inhibitor to block neutrophil superoxide production. Having confirmed that crude extracts of soybean do contain inhibitory factors which affect neutrophil superoxide production, we have resolved polypeptides in an ethanolic extract of soybean flour by anion and cation exchange chromatography and preparative polyacrylamide gel electrophoresis. Fractions have been assayed for protease inhibitory activity and inhibition of neutrophil superoxide production. We have found an inverse relation between these two inhibitory activities during the purification process. Two of three isolated polypeptides are potent inhibitors of neutrophil superoxide production (50% inhibition at 10(-7) M) but retain only weak anti-trypsin activity. A third polypeptide is a potent inhibitor of trypsin but is completely lacking superoxide inhibitory activity. None of the isolated polypeptides inhibit chymotrypsin. The implications of these findings for the hypothetical association between neutrophil production of superoxide and cellular proteases are discussed.