Bean Inhibitor Research Articles

Lima bean protease inhibitor: reduction and reoxidation of the disulfide bonds and their reactivity in the trypsin-inhibitor complex.

Lima bean protease inhibitor is a protein which inhibits both trypsin and chymotrypsin at different and independent sites. Complete reduction of the disulfide bonds of the inhibitor results in loss of biological activity. By air oxidation of the reduced inhibitor, full chymotrypsin inhibitory activity and up to 50% of the trypsin inhibitory activity can be regained; the rates at which these activities are regained were different. Attempts to obtain selective cleavage of one or a few disulfide bonds by carefully controlling reaction conditions were unsuccessful. All the disulfide bonds of lima bean inhibitor appear equally accessible to the reagent and with a less than twofold molar excess of dithioerythritol over the molarity of disulfide bonds, complete reduction was obtained; both inhibitory activities were equally sensitive to reduction and were lost as a linear function of the average number of disulfide bonds reduced and carboxymethylated. In contrast to this, the disulfide bonds of lima bean inhibitor are stabilized when the inhibitor is in the form of a molar complex with trypsin. Under these conditions, only one out of a possible eight disulfide bonds could be reduced in the inhibitor with up to a 10-fold molar excess of dithioerythritol. The modified inhibitor obtained after dissociation of reduced and carboxymethylated trypsin–inhibitor complex appeared fully active against both trypsin and chymotrypsin.

Derivative sspectroscopy applied to tyrosyl chromophores. Studies on ribonuclease, lima bean inhibitors, insulin, and pancreatic trypsin inhibitor.

ADVERTISEMENT RETURN TO ISSUEPREVArticleDerivative spectroscopy applied to tyrosyl chromophores. Ribonuclease, lima bean inhibitors, insulin, and pancreatic trypsin inhibitorJohn F. Brandts and Lawrence J. KaplanCite this: Biochemistry 1973, 12, 10, 2011–2024Publication Date (Print):May 1, 1973Publication History Published online1 May 2002Published inissue 1 May 1973https://doi.org/10.1021/bi00734a027RIGHTS & PERMISSIONSArticle Views143Altmetric-Citations119LEARN 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

Calorimetry of Some Trypsin-Trypsin Inhibitor Reactions

The heats of reaction of trypsin with soybean (Kunitz), ovomucoid, and lima bean inhibitors have been measured at pH 5.0 and 10° and 25°. All the reactions were endothermic, with ΔH ranging from 8.6 Cal per mole for the lima bean inhibitor to 15.3 Cal per mole for the soybean inhibitor. Equilibrium constants for the association were calculated from the dependence of residual tryptic activity upon added inhibitor. Free energy changes ranged from -9.8 to -12.7 Cal per mole, and ΔS varied from 48 to 79 cal per mole degree. The change in heat capacity, calculated from (ΔH298 - ΔH283)/15, varied from -263 to -442 cal per mole degree. The reactions are in general characterized by large negative ΔG°, moderate positive ΔH, large positive ΔS, and moderately large -ΔCp. The thermodynamic quantities for conversion of native soybean inhibitor to its state in which the arg 64-ile 65 peptide bond is cleaved were calculated as ΔG°298 = -1400 cal per mole, ΔH298 = -4040 cal per mole, ΔS298 = -8.8 cal per mole degree, and ΔCp = -55 cal per mole degree. The results for complex formation and for conversion of native soybean inhibitor to its hydrolyzed form are thought to indicate that interactions other than those directly involved in changes in covalent bonding are thermodynamically significant.

Studies on soybean trypsin inhibitors. IV. Complete amino acid sequence and the anti-proteinase sites of Bowman-Birk soybean proteinase inhibitor.

Bowman-Birk inhibitor was partially hydrolyzed with chymotrypsin [EC 3. 4. 4. 5] at pH 3.0, and the subsequent reduction, S-carboxymethylation and gel filtration of the product yielded two fragments. One fragment containing a methionine residue was further degraded into two fragments with cyanogen bromide. By the amino acid determination and the terminal sequence analysis of these three fragments as well as of the whole protein, the tryptic peptides were aligned into the complete amino acid sequence of the inhibitor. The anti-chymotrypsin and the anti-trypsin sites of the inhibitor were also identified by the limited hydrolysis with these proteinases. The sequence of this inhibitor was found to be very similar to that of lima bean inhibitor IV (Tan and Stevens, 1971), and consisted of two homologous peptide regions, one includes the inhibitory site for trypsin [EC 3. 4. 4. 4] and the other that for chymotrypsin. Comparison with other leguminosae inhibitors was also discussed.

The purification by affinity chromatography of a proteinase inhibitor binding species of anhydro-chymotrypsin

A species of anhydro-chymotrypsin which binds proteinase inhibitors was purified by affinity chromatography on a lima bean inhibitor-sepharose column. This species is indistinguishable from alpha chymotrypsin in its immunological reactivity with rabbit anti-PMS-chymotrypsin serum and its inhibitor binding capacity with potato inhibitor I. A non-inhibitor binding fraction (30%) of anhydro-chymotrypsin was not retained by the affinity column and possessed only weak immunological competence compared with the inhibitor binding species. Competition experiments between the inhibitor binding species of anhydro-chymotrypsin and chymotrypsin for potato inhibitor I or for lima bean inhibitor demonstrated that exchange occurs rapidly and stoichiometrically. The reversible exchange between chymotrypsin and the inhibitor binding species in the complexes with inhibitors indicates a covalent bond involving serine #195 of chymotrypsin does not contribute significantly to the free energy of complex formation.

Identification of the chymotrypsin-reactive site of the Bowman-Birk soybean inhibitor

End-group analysis of the Bowman-Birk inhibitor which had been exposed to catalytic levels of chymotrypsin (EC 3.4.4.5) at pH 3.8 indicated the cleavage of a Leu-Ser bond. Performic acid oxidation of the chymotrypsin-modified inhibitor yielded two fragments which could be separated by gel filtration on Sephadex G-25. From the end groups and amino acid composition of these two fragments, the chymotrypsin-reactive site was identified as Leu-48 and Ser-49. As in the case of the trypsin-reactive site, the sequence of amino acids in the vicinity of the chymotrypsin-reactive site of the Bowman-Birk inhibitor bears a high degree of homology with the corresponding site of the lima bean inhibitor.

Identification of the trypsin-reactive site of the Bowman-Birk soybean inhibitor

Succinylation of the 5 lysine residues of the Bowman-Birk inhibitor abolished its antitryptic activity without affecting its ability to inhibit chymotrypsin. Guanidination of the 5 lysine residues and modification of 1 of the 2 arginine residues, however, had no effect on either activity. Guanidination of the Bowman-Birk inhibitor-trypsin complex followed by dissociation of the complex revealed that one of the lysine residues of Bowman-Birk inhibitor had been protected from modification while in the complex. This particular lysine residue was identified as lysine-17 by isolation and characterization of two fragments from trypsin-modified Bowman-Birk inhibitor which had been reduced and alkylated. From end-group analysis of the trypsin-modified Bowman-Birk inhibitor and of the two fragments isolated therefrom, a limited sequence of amino acids in the immediate vicinity of the trypsin-sensitive bond of Bowman-Birk inhibitor was derived. This sequence was found to bear a striking degree of homology to the proposed antitrypsin site of the lima bean inhibitor.

Significance of direct and indirect kinin formation by plasmin in human plasma

Human plasma was treated with p-iodobenzoate to inactivate plasmin inhibitors. Incubation of such plasma with streptokinase generated plasmin activity, detectable by caseinolysis. Simultaneously plasma kinin was liberated. Both lima bean inhibitor which specifically inhibits plasmin, and ϵ-amino caproic acid which prevents plasmin activation suppressed the formation of kinin, indicating that plasmin was involved in the streptokinase-induced kinin liberation. Plasma heated to 61° at pH 4 still developed caseinolytic activity upon activation with streptokinase, but no kinin formed. Lima bean inhibitor added to non-heated plasma after treatment with p-iodobenzoate and streptokinase did not block kinin-forming activity. These findings demonstrate that plasmin formed kinin only indirectly by activating a heat labile plasma kininogenase. This enzyme has properties of kininogenase I, equal to classical plasma kallikrein. At concentrations corresponding to those found in iodobenzoate-treated, streptokinase-activated human plasma, purified human plasmin did not liberate kinin from human kininogen I. Only when added at considerably higher concentrations was a direct kinin formation seen. It is concluded that plasmin acts both as a plasma kallikrein activator and as a kininogenase proper. However, under physiological conditions it probably forms kinin only indirectly.

Isolation and properties of a proteinase inhibitor of navy beans

A crude preparation of proteinase inhibitors of navy beans was found by ion-exchange chromatography to contain at least two inhibitors. Particular attention was given to the purification of one of these by chromatographic and electrophoretic methods. Details of selected procedures for the isolation of the inhibitor identified as chromatographic component V are described. Preincubation of this inhibitor with trypsin at pH 3 prior to addition of a substrate at a pH favorable for substrate hydrolysis blocks the esterolytic but not the proteolytic activity of this enzyme. The differential effect of low pH preincubation is greater than with other inhibitors used for comparison. In contrast with the low pH preincubation effects obtained with the navy bean inhibitor and trypsin, no such differential effect on the proteolytic and esterolytic activities of α-chymotrypsin were observed. An altered electrophoretic pattern and nonhomogeneity of the navy bean inhibitor after low pH preincubation with trypsin is consistent with the hypothesis that alteration of the inhibitor accounts, in part, for its subsequent differential effect on the esterolytic and proteolytic activities of this enzyme. The constant apparent specific activity of the navy bean inhibitor with different lots of trypsin containing varying proportions of active and inactive enzyme suggests that this inhibitor, unlike others, reacts with inactive as well as active trypsin.

Amino acid sequence of lima bean protease inhibitor component IV. 2. Isolation and sequence determination of the chymotryptic peptides and the complete amino acid sequence.

Using ion exchange chromatography, gel filtration and paper electrophoresis eight peptides were isolated in pure form from a chymotryptic digest of lima bean inhibitor component IV. They account for 76 out of the 84 resiodues of the protein. Complete or partial sequnces of these peptides were determined using classical methods. This information, together with inforation obtained on the tryptic peptides and reported in the preceding paper allowed the deduction of the complete amino acid sequence of lima bean inhibitor IV. The results show that the original protein preparation was heterogeneous as reflected by the occurrence of conservative substitutions at positions 26, 37 and 39. A detailed examination of the sequence revealed the presence of a repetitive sequence with residues 50 through 61 clearly homologous to residues 23 through 34. Of added interest is the fact that the anti‐chymotrypsin site of lima bean inhibitor, as recently determined by Krahn and Stevns, was found to be located in one of the homologous regions. This has led us to speculated that the anti‐trypsin site could be located in the other homologous region and that this double‐headed inhibitor, with indpendent sites for trypsin and chymotrypsin, may have evolved by gene duplication.

Amino acid sequence of lima bean protease inhibitor component IV. 1. Isolation and sequence determination of the tryptic peptides.

Commmerical lima bean inhibitor was fractionated into four apparently homogeneous components as previously described by Jones, Moore and Stein in 1963.Reduced and alkylated component IV was hydrolyzed with tryspin and the resulting peptides were separated by ion exchange charomatography on Dowexs 50 X2 or gel filtration on Bio‐Gel P‐6. Where necessary, further purification of the peptides was carried out by paper chromatography, paper electrophoresis or a combination of both.Seven peptides were obtained in pure form and their compositions are reported here. The amino acid sequenceof six of these peptides was determined, using classical methods. When allowance is made for the occurence of two homologous peptides, presumably resulting from heterogeneity in the original protein preparation, the tryptic peptides isolated, account for the complete composition of the protein.In an attempt to obtain overlapping sequences the tryptic digest was also performed on the reduced, alkylated and guanidinated protein. Five tryptic peptides, including one containing homoarginine as the carboxy‐terminal residue, were isolated in pure form from the digest; their amino acid compositions are reported.

Immunological aspects of the binding of substrate analogues and polypeptide inhibitors to α-chymotrypsin

The effects of substate analogues and various polypeptide inhibitors on the immunological behavior of α-chymotrupsin have been observed by means of quantitative complement fixation. The interaction of α-chymotrypsin with substrate analogues produced a slight increase of the complement fixed over that fixed by α-chymotrypsin alone. The immunological effects of the interaction of α-chymotrypsin with polypeptide inhibitors were not uniform. Soybean and basic pancreatic inhibitors showed very little, in any, effect on complement fixation. An inhibitor isolated from the navy bean (phaseolus vulgaris) produced a shift of the complement fixation curve to the left with nearly double the maximum amount of complement fixed. Complement fixation by chymotrypsinogen with rabbit anti-α-chymotrypsin sera, was much greater than that produced by α-chymotrypsin itself. It is possible that some chymotrypsinogen was present in the α-chymotrypsin used for immunization. Both the substrate and the polypeptide inhibitors were per se without any effect either on the anti-α-chymotrypsin antibodies or on the lytic activity of the complement, or on the complement fixation produced by nonrelated immunological systems. A conformational change of the enzyme upon the interaction with substrate analogues and the navy bean inhibitor may explain this immunochemical behavior of α-chymotrypsin.

Mise en evidence de la formation d'un complexe ternaire entre trypsine, α 2-macroglobuline et inhibiteur basique de pancreas

The complex formed between trypsin and α 2-macroglobulin retains the whole hydrolytic activity for benzoylarginine- p-nitroanilide (DL BAPNA). We have observed that the inhibition of this activity is dependent on the inhibitor used. While soya bean inhibitor (MW = 20 000) is ineffective, the basic pancreatic inhibitor (MW = 6 500) progressively inhibits the complex. In this latter case, we have observed the formation of a very weakly active ternary compound by two methods: kinetic and fluorescence polarization. Such differences suggest either that the contact site of trypsin for binding is not the same for each inhibitor or that the steric hindrance by macroglobulin prevents large inhibitors from reaching the active site. The pancreatic inhibitor can reach this site but its affinity is lower for the complex than for free trypsin.

Inhibition of Human Trypsin, Plasmin, and Thrombin by Naturally Occurring Inhibitors of Proteolytic Enzymes

Human trypsin was strongly inhibited by three apparently unrelated inhibitors: lima bean trypsin inhibitor, bovine colostrum inhibitor, and bovine Kunitz pancreatic inhibitor. It was inhibited to varying degrees by Kunitz soybean inhibitor, soybean inhibitor AA, kidney bean inhibitor, black-eyed pea inhibitor, navy bean inhibitor, and quail ovomucoid. It was essentially uninhibited by bovine Kazal pancreatic inhibitor, porcine Kazal pancreatic inhibitor, potato inhibitor, chicken ovoinhibitor, and 10 avian ovomucoids including that of chicken. Many of these proteins strongly inhibit bovine trypsin. Thus, inhibition of bovine trypsin by naturally occurring inhibitors is no index of their activities against human trypsin. The activities of these inhibitors against human plasmin were generally similar to those against human trypsin, except that the activities of lima bean inhibitor and quail ovomucoid were comparatively weaker and the activity of the Kunitz soybean inhibitor was much stronger. The esterolytic activity of human thrombin was not affected by any of the inhibitors.

Proteolytic, esterase and kinin-releasing activities of some Soviet snake venoms

The casein-, p-toluene-sulfonyl- l-arginine methyl ester- and benzoyl- l-arginine amide-hydrolyzing as well as the kinin-releasing activities of the viperids Vipera lebetina, Vipera ursini and Echis carinatus, the crotalids Agkistrodon halys halys and Agkistrodon halys blomhoffii and the elapid Naja naja oxiana venoms were studied. Viperid venoms were shown to contain kinin-releasing enzyme, as previously reported for crotalid venoms. The effect of inhibitors of proteolytic enzymes, such as aminocaproic acid, soy bean trypsin inhibitor and Trasylol, was studied on the kinin-releasing activity of the venoms mentioned above. Only the kinin-releasing activity of V. lebetina venom was inhibited by soy bean inhibitor.

Proteinase production by a species of Cephalosporium.

An unidentified Cephalosporium species produced an extracellular proteinase when grown in a variety of fermentation media under submerged culture conditions. Maximal enzyme yields were obtained in a medium containing 2% corn meal, 1% soybean meal, and 0.5% CaCO(3) in tap water. Optimal proteinase production in this medium occurred within a 72- to 96-hr growth period. High enzyme yields were also attained with media in which cottonseed meal, Fermatein, Pharmamedia, or soybean-alpha-protein was substituted for the soybean meal. The substitution of these ingredients for the corn meal resulted in significantly decreased proteinase yields. The addition of minerals or vitamins to the corn meal-soybean meal fermentation medium failed to enhance proteinase production. The enzyme was most active in an alkaline environment; maximal caseinolysis occurred at pH 7.5, whereas pH 8.5 was optimal for either hemoglobin or beta-lactoglobulin hydrolysis. Enzymatic activity was also noted with either bovine albumin fraction V or soybean-alpha-protein substrates, whereas ovalbumin was not susceptible to enzymatic attack. The enzyme was stable within the pH range of 3.0 to 9.5 at 25 C for 2 hr, and at 5 C for 24 hr. The proteinase was stable upon heating for 10 min at 35 to 45 C, but it was totally inactivated at 70 C. The proteinase was unaffected by soybean inhibitor, partially inactivated by lima bean inhibitor, and completely inactivated by ovomucoid inhibitor.

Proteinase Production by a Species of Cephalosporium

An unidentified Cephalosporium species produced an extracellular proteinase when grown in a variety of fermentation media under submerged culture conditions. Maximal enzyme yields were obtained in a medium containing 2% corn meal, 1% soybean meal, and 0.5% CaCO(3) in tap water. Optimal proteinase production in this medium occurred within a 72- to 96-hr growth period. High enzyme yields were also attained with media in which cottonseed meal, Fermatein, Pharmamedia, or soybean-alpha-protein was substituted for the soybean meal. The substitution of these ingredients for the corn meal resulted in significantly decreased proteinase yields. The addition of minerals or vitamins to the corn meal-soybean meal fermentation medium failed to enhance proteinase production. The enzyme was most active in an alkaline environment; maximal caseinolysis occurred at pH 7.5, whereas pH 8.5 was optimal for either hemoglobin or beta-lactoglobulin hydrolysis. Enzymatic activity was also noted with either bovine albumin fraction V or soybean-alpha-protein substrates, whereas ovalbumin was not susceptible to enzymatic attack. The enzyme was stable within the pH range of 3.0 to 9.5 at 25 C for 2 hr, and at 5 C for 24 hr. The proteinase was stable upon heating for 10 min at 35 to 45 C, but it was totally inactivated at 70 C. The proteinase was unaffected by soybean inhibitor, partially inactivated by lima bean inhibitor, and completely inactivated by ovomucoid inhibitor.

A MODIFIED BEAN ROOT INITIATION TEST FOR AUXINS AND INHIBITORS

Removal of tissues above the cotyledonary node from leafy stem cuttings of Contender beans, after imbibition and prior to potting in root initiation tests, increased the sensitivity to growth inhibitors, and reduced the variation in control plants. Response to small amounts of auxin increased slightly and declined for larger amounts.

A possible role for the plasma kinins in pancreatitis and shock: Alan P. Thal ( Robert S. Marx Laboratories of the Dept. of Surgery, Wayne State Univ. School of Medicine, Detroit, Mich., U.S.A.)

The casein-, p-toluene-sulfonyl-l-arginine methyl ester- and benzoyl-l-arginine amide-hydrolyzing as well as the kinin-releasing activities of the viperids Vipera lebetina, Vipera ursini and Echis carinatus, the crotalids Agkistrodon halys halys and Agkistrodon halys blomhoffii and the elapid Naja naja oxiana venoms were studied. Viperid venoms were shown to contain kinin-releasing enzyme, as previously reported for crotalid venoms.The effect of inhibitors of proteolytic enzymes, such as aminocaproic acid, soy bean trypsin inhibitor and Trasylol, was studied on the kinin-releasing activity of the venoms mentioned above. Only the kinin-releasing activity of V. lebetina venom was inhibited by soy bean inhibitor.

Enhancement of fibrinolysis and antagonism of antiplasmin by reversible plasmin inhibitors

Fibrin clots exposed to mixtures of plasmin with appropriate levels of ϵ-amino-n-caproic acid, lima beam trypsin inhibitor, or soybean trypsin inhibitor underwent greater lysis, after washing and further incubation with buffer only, than did clots exposed to plasmin alone. The destruction of plasmin by antiplasmin was antagonized by the bean inhibitors, but was potentiated by ϵ-amino-n-caproic acid.