Soybean Bowman-Birk Inhibitor Research Articles

Nucleotide sequence of a cDNA encoding soybean Bowman-Birk proteinase inhibitor.

Nucleotide sequence of a cDNA encoding soybean Bowman-Birk proteinase inhibitor.

Trypsin inhibitory activity in some rapeseed genotypes

Trypsin inhibitory activity (TIA) was measured in six rapeseed cultivars of the double-high (d.h.),‘O’ and ‘OO’ genotypes. The mean TIA value for the d.h. and ‘O’ cultivars was 3.9 IU g −1, whereas that for the ‘OO’ cultivars was 6.9 IU g −1. The chromatographic profiles for gel filtration with Superose 12 HR 10/30-FPLC for all cultivars were characterized by three TIA peaks, corresponding to fractions 16, 18 and 21 in all trials. The three inhibitors isolated represented varying percentages of the total TIA: peak 1, 4–10%; peak II, 13–33% and peak Ill, 60–85%. Also noted was a different behaviour to heat denaturation for inhibitors I and II, which were completely thermolabile in the case of the d.h. and ‘O’ cultivars, whereas in the ‘OO’ cultivars, inhibitor II exhibited some thermostability. Inhibitors I and II have apparent M 4 s higher than that of inhibitor Ill, which shows an electrophoretic mobility similar to the Bowman-Birk soybean inhibitor and high thermostability. The results of this study indicate that selection for the ‘OO’ genotypes has favoured an increase in thermostable inhibitors at the expense of thermolabile ones.

Homology among trypsin/chymotrypsin inhibitors from red kidney bean, Brazilian pink bean, lima bean, and soybean

Three trypsin/chymotrypsin inhibitors [R(A), R(B2), R(C)] from red kidney bean (RKB; Phaseolus vulgaris var. Linden) and three [B(A), B(B), B(C)] from Brazilian pink bean (BPB; P. vulgaris var. Rosinha G2) inhibited one trypsin and one chymotrypsin/mol simultaneously. One RKB inhibitor [R(B1)] inhibited one trypsin/mol. Dissociation constants of trypsin- and chymotrypsin-inhibitor complexes ranged from 0.48 to 5.37 nM. High homology was shown among the seven inhibitors by tryptic and chymotryptic peptide maps by using anion-exchange and reverse-phase chromatographies. The inhibitors also showed significant homology with lima bean and soybean Bowman-Birk inhibitors but not with soybean Kunitz inhibitor. The N-terminal 16 amino acid sequence of inhibitor B(B) showed high homology with several Bowman-Birk inhibitors. A convenient peptide mapping procedure especially suitable for cysteine-rich proteinase inhibitors and analysis of peptide mapping data are described. The procedure and data analysis method provide an approach to assess homology among proteins without sequence data.

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of the soybean Bowman‐Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to bovine α‐chymotrypsin and bovine β‐trypsin. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C(p), F-T(p) and F-T(t), respectively) to bovine alpha-chymotrypsin (alpha-chymotrypsin) and bovine beta-trypsin (beta-trypsin) has been investigated. On the basis of Ka values, the proteinase inhibitor affinity can be arranged as follows: alpha-chymotrypsin: BBI approximately beta-trypsin:BBI approximately beta-trypsin:F-T(t) approximately beta-trypsin:F-T(p) much greater than alpha-chymotrypsin:F-C(p). F-C(p), F-T(p) and F-T(t) do not inhibit beta-trypsin and alpha-chymotrypsin action, respectively. On lowering the pH from 9.5 to 4.5, values of Ka for BBI, F-C(p), F-T(p) and/or F-T(t) binding to alpha-chymotrypsin and beta-trypsin decrease, thus reflecting the acid-pK shift of the invariant His57 catalytic residue from 7.0, in the free enzymes, to 5.2, in the proteinase:inhibitor complexes. Considering the known molecular models, the observed binding behaviour of BBI, F-C(p), F-T(p) and F-T(t) was related to the inferred stereochemistry of the proteinase:inhibitor contact regions.

Effect of the reactant mixing sequence on the chymotrypsin inhibition assay

In assaying chymotrypsin inhibition by the soybean Bowman-Birk inhibitor, two sequences of mixing the reactants were tried: adding the substrate last (s-last test) or adding the enzyme last (e-last test). The inhibition values obtained from the s-last test were either equal to or lower than those from the e-last test, depending on the pre-mix pH and pre-incubation time, while the values from the e-last test were independent of these conditions. The differences between the two tests were largest at a pre-mix pH of 4.0 or 8.5 and zero near pH 7.0. Abruptly changing the pre-mix pH from 4.0 to 7.0 brought the values of the two tests closer. These observations suggest a reversible, yet limited, hydrolysis of the inhibitor by the very enzyme it inhibits and indicate the greater reliability of the e-last test over the s-last test, paralleling a similar previous finding on the trypsin inhibition assay.

Molecular cloning and analysis of four potato tuber mRNAs

Tuberization in potato is a complex developmental process involving the expression of a specific set of genes leading to the synthesis of tuber proteins. We here report the cloning and analysis of mRNAs encoding tuber proteins. From a potato tuber cDNA library four different recombinants were isolated which hybridized predominantly with tuber mRNAs. Northern blot hybridization experiments showed that three of them, pPATB2, p303 and p340, can be regarded as tuber-specific while the fourth, p322, hybridizes to tuber and stem mRNA. Hybrid-selected in vitro translation and nucleotide sequence analysis indicate that pPATB2 and p303 represent patatin and the proteinase inhibitor II mRNA respectively. Recombinant p322 represents an mRNA encoding a polypeptide having homology with the soybean Bowman-Birk proteinase inhibitor while p340 represents an mRNA encoding a polypeptide showing homology with the winged bean Kunitz trypsin inhibitor. In total, these four polypeptides constitute approximately 50% of the soluble tuber protein. Using Southern blot analysis of potato DNA we estimate that these mRNAs are encoded by small multigene families.

Anti-chymotrypsin and anti-elastase activities of a synthetic bicyclic fragment containing a chymotrypsin-reactive site of soybean Bowman-Birk inhibitor

A bicyclic hexadecapeptide, which corresponds to the sequence 36–51 and contains the chymotrypsin-reactive Leu-43-Ser-44 bond of soybean Bowman-Birk inhibitor, has been synthesized. This peptide consists of two loops formed by disulfide bridges between Cys-36 and Cys-51 and between Cys-41 and Cys-49. The bicyclic peptide showed a strong anti-cymotryptic activity with a K i of 7.1 · 10 −7 M. Comparison of inhibitory activity and digestive stability against chymotrypsin with other hexadecapeptides having the same sequence but lacking one or both disulfide bridges suggested that the compact bicyclic structure increases the activity and protects the Leu-Ser bond from chymotryptic digestion. Interestingly, the bicyclic peptide was found to inhibit porcine pancreatic elastase with a K i of 4.3 · 10 −5 M, indicating the broad specificity of this ring system.

The complete amino acid sequence of rice bran trypsin inhibitor.

The complete amino acid sequence of a double-headed trypsin inhibitor (RBTI) from rice bran was determined by a combination of limited proteolysis of the native inhibitor with Streptomyces griseus trypsin at pH 3 and conventional methods. RBTI consists of 133 amino acid residues including 18 half-cystine residues which are involved in 9 disulfide bridges in the molecule. The limited proteolysis at pH 3 produced a major split of Lys(83)-Met(84) and a minor split of Arg(107)-Val(108) together with a non-enzymatic hydrolysis of Asp(19)-Pro(20) in the molecule. The established sequence showed that RBTI is composed of 4 domains, domains I and III, and domains II and IV being homologous to the first and the second domains of soybean Bowman-Birk inhibitor, respectively, indicating that RBTI has a duplicated structure of the Bowman-Birk type inhibitor.

Proteolysis of soybean bowman-birk trypsin inhibitor during germination,

Proteolysis of soybean bowman-birk trypsin inhibitor during germination,

Proteolysis of soybean bowman-birk trypsin inhibitor during germination

The Bowman—Birk type trypsin inhibitor, BBSTI-D, which appears in the cotyledons of germinated soybeans ( Glycine max), was isolated in homogeneous form. BBSTI-D has an amino acid composition identical to the native Bowman—Birk soybean trypsin inhibitor (BBSTI-E) except for the loss of one glutamyl/glutaminyl residue and one aspartyl/asparaginyl residue. The amino-terminal sequence of BBSTI-D was identical to that of BBSTI-E. These data, as well as the compositions of the tryptic peptides from reduced carboxymethylated BBSTI-D, indicate that BBSTI-D is derived from BBSTI-E by the loss of the carboxyl-terminal residues Glu 70—Asn 71.

Ultrastructural localization of bowman-birk inhibitor on thin sections of Glycine max (Soybean) cv. Maple Arrow by the gold method

The soybean Bowman-Birk inhibitor (BBI), a polypeptide of MW 8,000, has a specificity directed against trypsin and chymotrypsin. BBI was localized at the ultrastructural level by the protein A gold method on thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledon and embryonic axis, BBI was found in all protein bodies, the nucleus and, to a lesser extent, the cytoplasm. Contrary to the Kunitz trypsin inhibitor (Horisberger and Tacchini-Vonlanthen 1983), BBI was not present in the cell wall but was found in the intercellular space. Intensity of marking in cotyledons of four-day-old seedlings was similar with the exception of the intercellular space which was free of BBI. In two lines lacking the Kunitz inhibitor (P.I. 157440 and 196168), data indicated that marking intensity was similar to that of cv. Maple Arrow. In contrast, in varieties lacking the lectin (Norredo, T-102) marking was more intense than in cv. Maple Arrow.

Anti-tryptic activity of a synthetic bicyclic fragment of soybean bowman-birk inhibitor

A bicyclic hexadecapeptide corresponding to the sequence 9–24 of soybean Bowman-Birk inhibitor has been synthesized. This peptide has two disulfide linkages, between residues 9 and 24 and between 14 and 22. A nonapeptide loop formed by the latter disulfide bridge contains a Lys-16-Ser-17 bond, which is the reactive site for trypsin inhibition. The bicyclic peptide showed strong anti-tryptic activity with an inhibition constant ( K i) of 1.5·10 −7 M. Comparison with other hexadecapeptides which have the same sequence but lack one or both disulfides suggested that a compact ring system increases the activity and protects the Lys-Ser bond from tryptic hydrolysis.

Alkaline proteases in digestive juice of the silkworm, Bombyx mori

Two alkaline proteases existing in the digestive juice of the fifth larval instar of the silkworm, Bombyx mori, were isolated by using Amberlite IRC-50 ion-exchange chromatography and soybean Bowman-Birk inhibitorSepharose 4B affinity chromatography. One named P-II was not retained with Amberlite IRC-50, but adsorbed to soybean Bowman-Birk inhibitor-Sepharose 4B and further separated into three species, P-IIa, P-IIb and P-IIc by Bio-Rex 70. Another one named P-III was retained with Amberlite IRC-50 and adsorbed to soybean Bowman-Birk inhibitor-Sepharose 4B and also further separated into three species, P-IIIa, P-IIIb and P-IIIc by Bio-Rex 70. Each species belonging to the same protease group had nearly identical characters. The molecular weights of P-II and P-III were 22000 and 23000, respectively. The amino acid compositions of P-II and P-III were clearly different from each other, but the characteristics of amino acid distribution were similar. The P-II had its pH optimum at about pH 10, and couldhydrolyze benzoylarginine- p-nitroanilide (Bz-Arg-pNA) and was inhibited by diisopropyl fluorophosphate, TLCK, leupeptin, antipain, HgCl 2 and soybean Bowman-Birk inhibitor. These facts suggest that the P-II should belong to trypsin-like protease. On the other hand, the P-III had its pH optimum at about pH 11, and was inhibited by diisopropyl fluorophosphate, chymostatin and soybean Bowman-Birk inhibitor, but not by TPCK, and moreover it could not hydrolyze Bz-Arg-pNA, Bz-Tyr-pNA or glutaryl- l- phenylalanine-p- nitroanilide (Glt-Phe-pNA). The active-site character and substrate specificity of P-III are not clear at present.

Chemical substitutions of the reactive site leucine residue in soybean Bowman-Birk proteinase inhibitor with other amino acids.

A method of exchanging amino acid residue (P1 residue) which determines primarily the inhibitory specificity of a proteinase inhibitor is described. Leu43 (P1 residue) at the chymotrypsin reactive site of Bowman-Birk soybean inhibitor was removed by limited proteolysis of Leu43-Ser44 bond followed by carboxypeptidase digestion. An appropriate amino acid methyl ester was introduced into this position by water-soluble carbodiimide. Derivatives having methyl esters of Gly, Ala, Val, Met, Leu, Phe, Trp, or D-Trp at position 43 were prepared and were shown to restore the activity to various extents. This method provides new approach to study structure-function relationship of proteinase inhibitors and to prepare novel inhibitors.

Circular dichroism spectra of cleavage fragments of soybean trypsin-chymotrypsin inhibitor.

Circular dichroism spectra of biologically active fragments of Bowman-Birk soybean inhibitor have been determined in acidic, neutral, and alkaline conditions. Neither fragment showed evidence of alpha-helix or beta-structure. Negative dichroism above 260nm has been assigned in both fragments primarily to disulfide bonds, with a minor contribution from tyrosine in a hydrophilic environment. The individual spectra of these fragments, and their sum between 230 and 340 nm have been compared with the spectra of the intact inhibitor and several structurally related proteins. Possible interactions which may give rise to CD bands in this region are discussed.

Isolation and some properties of two fragments with inhibitory activity obtained from adzuki bean proteinase inhibitor by peptic digestion.

Proteinase inhibitor II' from adzuki beans was subjected to peptic digestion. One of the resulting fragments, which inhibited chymotrypsin but not trypsin, was composed of 27 amino acid residues. The fragment was confirmed to be derived from the chymotrypsin-inhibitory domain of the original inhibitor. Another fragment, which inhibited trypsin only, contained 38 amino acid residues and consisted of two peptide chains. One of them, consisting of 25 amino acid residues, corresponded to the original reactive site region for trypsin. These fragments were also obtained from inhibitor II by peptic digestion. These findings, confirm that these inhibitors, which do not inhibit chymotrypsin and trypsin simultaneously, have separate and independent domains for the inhibition of each enzyme. The active fragments are homologous in chemical structures with the two fragments from soybean Bowman-Birk proteinase inhibitor. However, unlike the fragments from Bowman-Birk inhibitor, our chymotrypsin-inhibitory fragment was a potent inhibitor of the enzyme and was as resistant as the intact inhibitor to the attack of excess chymotrypsin. The trypsin-inhibitory fragment had a lower anti-tryptic action than the original inhibitor and was gradually inactivated by trypsin. These differences between our fragments and those of the Bowman-Birk inhibitor are probably a result of the replacement of a few amino acid residues in the reactive site regions.

The amino acid sequence of adzuki bean proteinase inhibitor I

1. Introduction Proteins inhibiting the activity of proteinases are widely distributed in plant and animal tissues, and they are especially rich in leguminous seeds [ 11. Knowledge of the amino acid sequences of these proteinase inhibitors is important for studies on their structure- function relationships and molecular evolution. As for legume inhibitors, the amino acid sequences of Bowman-Birk soybean inhibitor [2] and two addi- tional soybean inhibitors [3] have been determined completely, and those of lima bean inhibitors IV and IV’ [4] and garden bean inhibitors II and II’ [S] nearly completely. These results show that these legume inhibitors are a family of low molecular weight proteins composed of about 70-80 amino acid residues and are double-headed, i.e., consisting of two domains each having a proteinase reactive site. Here, we report the amino acid sequence of adzuki bean proteinase inhibitor I, one of the major proteinase inhibitors isolated from adzuki bean (Phase&s

Inhibitory properties of nonapeptide loop structures related to reactive sites of soybean Bowman-Birk inhibitor

Inhibitory properties of nonapeptide loop structures related to reactive sites of soybean Bowman-Birk inhibitor

Studies on soybean trypsin inhibitors. XIII. Preparation and characterization of active fragments from Bowman-Birk proteinase inhibitor.

Soybean Bowman-Birk inhibitor, a double-headed inhibitor of trypsin and alpha-chymotrypsin, was treated with cyanogen bromide and then pepsin to yield two inhibitory active fragments. Structural investigation showed that one of the fragments was derived from the trypsin inhibitory domain and the other from the chymotrypsin inhibitory domain of the inhibitor. In contrast to the unusual stability of the native inhibitor, the separated domains were less stable and could be inactivated with excess proteinases. These results suggest that the legume double-headed inhibitors acquired their unusual stability by duplicating an ancestral single-headed structure.