Purification and Properties of the Trypsin Inhibitors from Buckwheat Seed

The trypsin inhibitors in buckwheat seeds were isolated by affinity chromatography on trypsin-Sepharose 4B, and the components were fractionated by chromatography on DEAE-Sepharose CL-6B. The major components, inhibitors I, II and III, were found to be homogeneous proteins with molecular weight of about 8,000. Trypsin inhibitory activity was more pronounced than the chymotrypsin inhibitory activity in all the inhibitor preparation obtained. The three major inhibitors had similar amino acid compositions and had no detectable amounts of tryptophan and carbohydrate. A high level of acidic and basic amino acid residues and a low level of methionine, tyrosine and phenylalanine residues characterized the inhibitors. Although the inhibitors I and II were particularly thermostable, inhibitor III, the most abundant component, was shown to be relatively heat-labile.     

Heterogeneity of the basic pancreatic inhibitor (Kunitz) in various bovine organs

Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10 600-6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion-exchange chromatography; they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7-4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross-reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test.     

Trypsin inhibitors in summer squash (Cucurbita pepo) seeds. Isolation, purification and partial characterization of three inhibitors

Three trypsin inhibitors were isolated from summer squash (Cucurbita pepo) seeds and purified to homogeneity by fractionation with ammonium sulphate and methanol, ion-exchange chromatography and gel filtration. All three inhibitors have lysine at their active site. Two of them (II, IV) show the same isoelectric point (at pH 5.6), amino acid composition and molecular mass (3259). The third inhibitor (III) of molecular mass of 3654 and isoelectric point at 4.9 has additionally one histidine residue and two glutamic acid residues more per molecule.     

Isolation of a trypsin-like enzyme from Streptomyces paromomycinus (paromotrypsin) by affinity adsorption through Kunitz inhibitor-sepharose.

A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 mu mg-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000-25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Argo-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhbited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.     

Purification and characterization of proteinase inhibitors from winged bean (Psophocarpus tetragonolobus (L.) DC.) seeds

Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.     

Chymotrypsin inhibitors from hemolymph of the silkworm, Bombyx mori.

Three new protease inhibitors were isolated and purified about 200-fold from hemolymph of silkworm larvae, Bombyx mori, using ion-exchange and affinity chromatography. Two of the three inhibitors were basic proteins (SCI-I had pI 9.4 and SCI-II had pI 9.6) and one was acidic (SCI-III had pI 4.0). The molecular weight of each inhibitor was determined to be 7,000 by the sedimentation equilibrium method. The amino acid composition of the inhibitors were similar except for the contents of Asp, Glu, Ile, Leu, and Lys. Val, His, and Trp were not present in the inhibitors and Met appeared only in SCI-III. The CD spectra of the inhibitors were all similar and indicated a low content of alpha-helical structure (10% at most). Each inhibitor could inhibit the protease and esterase activities of bovine alpha-chymotrypsin at a one-to-one molar ratio, and the dissociation constants were 3.1 X 10(-9)M for SCI-I and II and 1.3 X 10(-8)M for SCI-III. Only SCI-II showed a weak inhibitory activity against bovine trypsin. Subtilisin BPN' and papain were not inhibited by these inhibitors.     

The Complete Amino Acid Sequence of a Trypsin Inhibitor from Bauhinia variegata var. candida Seeds

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M _r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K _i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64).     

The Complete Amino Acid Sequence of a Trypsin Inhibitor from Bauhinia variegata var. candida Seeds

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M _r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K _i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64).     

Isolation and partial characterization of the pancreatic secretory trypsin inhibitor in the rat

Isolation in a 55% yield of the low molecular weight pancreatic secretory trypsin inhibitor was achieved by gel filtration of an acid extract of whole inactive rat pancreas juice on Sephadex G-50 at pH 2.5 followed by desalting and ion-exchange chromatography on SP Sephadex C-50 at pH 4.5. Two distinct chromatographic fractions were obtained, labeled fraction 1 and 2. Fractions 1 and 2 showed three, respectively two, distinct closely migrating cationic bands on gel electrophoresis in barbital buffer, pH 8.6. Each fraction demonstrated one band on polyacrylamide disc electrophoresis at pH 4.6. The inhibitor is homogenous on gel filtration and on the basis of its stoichiometry with active site titrated rat anionic trypsin. Its molecular weight is approx. 6024. The amino acid composition is included. Rat pancreatic secretory trypsin inhibitor is trypsin-specific and interacts on a 1:1 molar basis with rat trypsin. It is good inhibitor of bovine trypsin but a poor inhibitor of human cationic trypsin and its binding to trypsin is reversible by acidification. Like other inhibitors of this sort, it is present in about 0.1–0.2% of the total protein content of the juice, and normally exists in its free form. A simple procedure for the production of antiserum to the inhibitor which is a poor antigen is also described.     

Isolation and primary structure of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds.

The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities. The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M(r) 19,242 and M(r) 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.     

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.

A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.     

Purification and some properties of two proteinase inhibitors (DE-1 and DE-3) from Erythrina latissima (broad-leaved erythrina) seed

Two proteinase inhibitors, DE-1 and DE-3, were purified from Erythrina latissima seeds. Whereas DE-1 inhibits bovine chymotrypsin and not bovine trypsin, DE-3 inhibits trypsin but not chymotrypsin. The molecular weights and the amino acid compositions of the two inhibitors resemble the corresponding properties of the Kunitz-type proteinase inhibitors. The N-terminal primary structure of DE-3 showed homology with soybean trypsin inhibitor (Kunitz) and also with the proteinase inhibitors (A-II and B-II) from Albizzia julibrissin seed.     

Isolation and characterization of a new trypsin inhibitor from Crotalaria paulina seeds

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a Ki value of 4.5 x 10(-8) M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors.     

Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type

A number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr = 14,500) and II (Mr = 7,000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors II are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.     

Bovine pancreatic trypsin inhibitor and homologous polypeptide inhibitors in nephron cells.

Bovine pancreatic trypsin inhibitor (BPTI, aprotinin) is a fifty-eight amino acid polypeptide, which is present together with related molecular isoforms in various bovine organs. In the present study these protease inhibitors were isolated from bovine kidney by affinity chromatography on immobilized trypsin and a subsequent FPLC step. Due to their electrophoretic, structural, and inhibitory properties, the inhibitors were strictly similar to the polypeptides identified previously in other bovine organs. Immunohistochemical experiments showed a widespread localization of these polypeptides in nephron epithelial cells (proximal and distal tubules, loop of Henle, collecting tubules).     

Inhibition of rat and bovine trypsins and chymotrypsins by soybean, bovine basic pancreatic, and bovine colostrum trypsin inhibitors.

1. Bovine (Bos taurus) trypsin and trypsin activity in rat (Rattus norvegicus) pancreatic extract were inhibited by soybean trypsin inhibitor and by bovine basic pancreatic and colostrum inhibitors. 2. Bovine alpha-chymotrypsin was inhibited by soybean and bovine basic pancreatic inhibitors but only weakly by colostrum inhibitor. 3. Chymotrypsin activity in rat pancreatic extract was due to at least three different components against all of which the inhibitors were largely ineffective. 4. It is concluded that bovine colostrum inhibitor has a more limited inhibition spectrum than the phylogenetically related basic pancreatic inhibitor which, in turn, is less active against rat than against bovine enzymes.     

A new inhibitor of plasmin and trypsin from porcine leukocytes.

A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.     

Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.     

Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz).     

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.