Complete Amino Acid Sequences of Three Proteinase Inhibitors from White Sword Bean (Canavalia gladiata)

Three major serine proteinase inhibitors (SBI-1, -2, and -3) were purified from the seeds of white sword bean (Canavalia gladiata) by FPLC and reversed-phase HPLC. The sequences of these inhibitors were established by automatic Edman degradation and TOF-mass spectrometry. SBI-1, -2, and -3 consisted of 72, 73, and 75 amino acid residues, with molecular masses of 7806.5, 7919.8, and 8163.4, respectively. The sequences of SBI-1 and -2 coincided with those of CLT I and II [Terada et al. (1994) Biosci. Biotech. Biochem., 58, 376-379] except only N- or C-terminal amino acid residues. Analysis of the amino acid sequences showed that the active sites of the inhibitors contained a Lys²¹-Ser²² against trypsin and Leu⁴⁸-Ser⁴⁹ against chymotrypsin, respectively. Further, it became apparent that about seven disulfide bonds were present. These results suggest that sword bean inhibitors are members of the Bowman-Birk proteinase inhibitor family.     

The reactive site of eggplant trypsin inhibitor.

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.     

Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis).

Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule.     

Trypsin reactivity site of the Vicia angustifolia proteinase inhibitor

Trypsin [EC 3.4.21.4] modified (reactive site cleaved) Vicia angustifolia proteinase inhibitor was prepared at pH 3 with a catalytic amount of trypsin and purified using columns of Sephadex G-50 and DEAE-Sephadex A-25. The modified inhibitor, which still retained antitryptic activity, lost its activity upon treatment with carboxypeptidase B or citraconic anhydride. End-group analyses revealed that the carboxyl-terminal Arg and the amino-terminal Ser residues were newly exposed end-groups in the modified inhibitor. It takes a much longer incubation time (about 1 h) to exhibit the maximal inhibitory activity against trypsin. Reduction and carboxymethylation of the modified inhibitor produced two fragments on Sephadex G-50 chromatography. The smaller fragment consisted of about 32 amino acid residues and possessed a new carboxyl-terminal Arg residue. The larger fragment consisted of about 80 residues and possessed a Ser residue at its amino-terminus. These results indicate that the small fragment was derived from the amino-terminal portion of the modified inhibitor and the large fragment from the carboxyl-terminal. It is also concluded that an Arg-Ser bond is the reactive site as well as the inhibitory site of the V. angustifolia inhibitor against trypsin. The sequence around the antitryptic site exhibits high degrees of homology with other double-headed inhibitors of legume origin, such as the Bowman-Birk inhibitor, lima beam inhibitor, and the major inhibitor in chick-peas.     

The release of proteinase inhibitors from legume seeds during germination

The seeds of twelve common species of legumes were examined for the release of proteinase inhibitor activity during germination. All species released inhibitory activity against bovine trypsin (EC 3.4.21.4), ranging from 1.0 unit per g dry wt. of seed in 24 hr for soybean (Glycine max), to 0.07 unit per g for broad beans (Vicia faba) and sugar pod peas (Pisum sativum). This release corresponds to approximately 1–13 % of the total trypsin inhibitory activity of the seed, with lentils (Lens culinaris) releasing the greatest percentage, and the scarlet runner bean (Phaseolus coccineus) the least. In most species the amount of inhibitor released increases until 24–48 hr of germination, and then remains roughly the same or decreases slightly by 72 hr of germination. Five species of legumes were also examined for the release of inhibitory activity against bovine chymotrypsin (EC 3.4.21.1). In each case chymotryptic inhibitory activity was released in a manner similar to the trypsin inhibitor.     

Temporary site in the anti-tryptic fragment from adzuki-bean proteinase inhibitor II

The anti-tryptic fragment, derived from adzuki-bean proteinase inhibitor II, was subjected to limited proteolysis by trypsin at pH 2.9 for 48 h. Three peptide bonds, Lys-Ser, Arg-Cys and Arg-Asp, were split, inactivating the fragment. The temporary site, the point of inactivation against trypsin, was concluded to be Arg-Cys, since the Lys-Ser bond is the reactive site and the tripeptide (Asp)_3′ released by the cleavage of the Arg-Asp bond, should not affect the inhibitory activity. This effective bond, corresponding to Arg³²-Cys³³ of inhibitor II, was possibly more exposed to the enviromental solvent by cuting down the anti-chymotryptic domain from the parent inhibitor.     

Protective effect of the pteroylpolyglutamates and phosphate on the proteolytic inactivation of thymidylate synthetase

Thymidylate synthetase is readily inactivated by trypsin, chymotrypsin, and carboxypeptidase A when incubated in 10–20 mm potassium phosphate buffer (pH 7.0). The loss is activity produced by trypsin and chymotrypsin is accomplished by extensive protein degradation, while inactivation by carboxypeptidase A is accompanied by release of the carboxyl-terminal valine only (Aull et al., 1974, J. Biol. Chem., 249, 1167–1172). In contrast, when the incubations are conducted in 100–200 mm potassium phosphate buffer (pH 7.0), the synthetase is not inactivated by any of the three enzymes and the results of amino acid analysis and sodium dodecyl sulfate disc gel electrophoresis demonstrate that proteolysis is prevented. The resistance of thymidylate synthetase to inactivation was shown not to be due to the inhibition of the proteolytic enzymes by the buffer. The inactivation is not prevented either by pteroylmonoglutamates or by 2′-deoxyuridine 5′-phosphate (dUMP) alone, but the presence of both is partially protective. The pteroylpolyglutamates, however, offer limited protection against carboxypeptidase A and chymotrypsin; in combination with dUMP, proteolytic inactivation of the snythetase by all three enzymes is prevented. Characterization of the properties of carboxypeptidase A-inactivated thymidylate synthetase reveals the following, (i) The binding of deoxynucleotides is unaltered, but the binding of a variety of pteroylpolyglutamate derivatives is reduced or abolished, (ii) Pteroylpolyglutamates are bound provided dUMP or an analog such as 5-fluorodUMP is present, (iii) Ternary complex formation between carboxypeptidase A-inactivated enzyme and (+)5,10-methylenetetrahydropteroyltetraglutamate plus 5-fluorodUMP occurs in the same molar binding ratio (1:2:2) at saturation as with the native enzyme, but differs from the native enzyme ternary complex in that the dissociation constant for 5-fluorodUMP is increased by approximately 10⁵. In addition, there is no evidence for the formation of covalent linkages between the ligands and enzyme, (iv) The treated enzyme cannot catalyze tritium release from [³H₅]dUMP in the presence of either (+)5,10-methylenepteroylmonoglutamate or (+)5,10-methylenetetrahydropteroyltetraglutamate.     

Purification and Characterization of a Proteinase Inhibitor from Field Bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^?1 and 3.1 × 10⁷ M^?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Inhibition mechanism of a peanut trypsin-chymotrypsin inhibitor, B-III: determination of the reactive sites for trypsin and chymotrypsin

Peanut inhibitor B-III was found to form two types of complexes with trypsin, T2I and TI, by gel filtration HPLC. Two cleaved peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in the trypsin modified inhibitor (TM-B-III*R*S) (J. Biochem. 93, 479-485 (1983] were resynthesized by the complex formation with 2 mol of trypsin. These results suggest that the two peptide bonds may be the reactive sites for trypsin. TM-B-III*R*S inhibited bovine trypsin as well as native B-III but had little chymotrypsin inhibitory activity. The two peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in B-III were cleaved partly by prolonged incubation with a catalytic amount of chymotrypsin. But gel filtration HPLC of the chymotrypsin-inhibitor complex showed the formation of only CI complex. Incubation of TM-B-III*R*S with an equimolar amount of chymotrypsin resulted in the resynthesis of only the Arg(10)-Arg(11) bond. These findings suggest that Arg(10)-Arg(11) may be a true reactive site for chymotrypsin. An inhibition mechanism of B-III against trypsin and chymotrypsin was proposed from the results obtained by the present studies.     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Comparative studies on S-adenosyl-l-methionine binding sites of protein N-methyltransferases,using 8-azido-S-adenosyl-l-methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Purification and characterization of the western spruce budworm larval midgut proteinases and comparison of gut activities of laboratory-reared and field-collected insects.

Three proteolytic enzymes, trypsin, chymotrypsin, and aminopeptidase-N (APN), were purified from laboratory-reared western spruce budworm, Choristoneura occidentalis [Freeman], larvae. Budworm trypsin exhibited a high degree of substrate specificity, was inactivated by DFP and TLCK, and was inhibited by trypsin inhibitors. The western spruce budworm chymotrypsin hydrolyzed SAAPFpNA and SAAPLpNA, but not SFpNA, SGGFpNA, SGGLpNA or BTpNA. The chymotrypsin was inactivated by DFP, and was inhibited by chymostatin and the chymotrypsin inhibitor, POT-1. Purified budworm chymotrypsin exhibited little BTEE esterolytic activity and was insensitive to inhibition with TPCK. The N-terminal sequence of budworm trypsin, chymotrypsin, and APN were obtained. Similar levels of trypsin and APN gut activities were found in laboratory-reared and field-collected larvae. However, in comparison to laboratory-reared insects, considerably less chymotrypsin activity, and a much higher level of gut carboxypeptidase activity were found in field-collected western spruce budworm larvae.     

Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^–1 and 3.1 × 10⁷ M^–1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Purification and characterization of an acidic trypsin/subtilisin inhibitor from tortoise egg white

Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11 700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100°C for 4 h at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation contants are 7.2·10^?9 M for bovine trypsin adn 5.5·10^?7 M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

The amino acid sequence of the double-headed protein proteinase inhibitor from dog submandibular glands, I. Structural homology to the pancreatic secretory trypsin inhibitors (author's transl)]

The primary structure of the broad specificity proteinase inhibitor from dog submandibular glands was elucidated. The inhibitor consists of a single polypeptide chain of 117 amino acids which is folded into two domains (heads) connected by a peptide of three amino acid residues. Both domains I and II show a clear structural homology to each other as well as to the single-headed pancreatic secretory trypsin inhibitors (Kazal type). The trypsin reactive site (-Cys-Pro-Arg-Leu-His-Glx-Pro-Ile-Cys-) is located in domain I and the chymotrypsin reactive center (-Cys-Thr-Met-Asp-Tyr-Asx-Arg-Pro-Leu-Tyr-Cys-) in domain II, cf. the Figure. The inhibitor is thus double-headed with two independent reactive sites. Whereas head I is responsible for the inhibition of trypsin and plasmin, head II is responsible for the inhibition of chymotrypsin, subtilisin, elastase and probably also Aspergillus oryzae protease and pronase. Remarkably, the structural homology exists also to the single-headed acrosin-trypsin inhibitors from seminal plasma[12] and the Japanese quail inhibitor composed of three domains[13].     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Identification of a methionine residue as the reactive site for chymotrypsin in the double-headed proteinase inhibitor from the canine submandibular gland (author's transl)]

The canine submandibular inhibitor is double-headed with two independent reactive sites. Whereas the trypsin-reactive center (-Ala-Cys-Pro-Arg26-Leu-His-) is located in domain I, the chymotrypsin-reactive site (-Met-Cys-Thr-Met78-Asp-Tyr-) is located in domain II. The presence of a methionine residue in this inhibition center is supported by the findings that nitration with tetranitromethane abolishes neither trypsin nor chymotrypsin inhibition, whereas after alkylation of the methione residues, only trypsin inhibition is retained. Remarkably, another inhibitor from microbial sources [10] which also contains a methionine residue in the presumed reactive site also inhibits subtilisin but not chymotrypsin (or trypsin).     

Further confirmation of carboxypeptidase Y as a metal-free enzyme having a reactive serine residue.

The metal content of carboxypeptidase Y was analyzed by the atomic absorption method. After exhaustive dialysis against an EDTA solution, the enzyme showed no loss of activity nor any significant content of metals (Zh,Mg,Ca,Cu,Mn,Ni,Fe, and Co). The activity was, however, rather sensitive to preincubation with various metals. The reactivity of a serine residue of the enzyme was also reevaluated. Diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) stoichiometrically and irreversively inhibited the enzyme. The rate of inactivation with DFP was much faster than that for typsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1.], while the rate with PMSF was one-fifteenth of that for chymotrypsin. The pH-dependence of the inactivation by DFP was similar to that of the enzymatic hydrolysis of acetylphenylalanine ethyl ester. The present results indicate that carboxypeptidase Y is free of metals and has a serine residue with a vital role in the catalytic process, though the functional role of this SH group remains to be clarified.