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.     

Broad-specificity proteinase inhibitors in Scopolia japonica (Solanaceae) cultured cells. Isolation, physicochemical properties, and inhibition kinetics.

Proteinase inhibitors were isolated from Scopolia japonica cultured cells. Isolation procedures involve concentration by a hydrophobic resin of Diaion HP-20, decolorization by Duolite A-7, affinity chromatography on trypsin-Sepharose, and Bio-Gel P-4 chromatography. It was found that the proteinase inhibitors from S. japonica cells are a mixture of at least five components. For the inhibitory components except one, amino acid analyses, measurements of sedimentation equilibrium and optical rotatory dispersion (ORD) were carried out. The inhibitors were shown to be the polypeptides with molecular weights in the range of approximately 4000 to 6000. In addition, one of them was found to have approximately 15% alpha-helical conformation by the Moffitt-Yang analysis of ORD data. The inhibitors were found to have potent inhibitory activity for trypsin, chymotrypsin, plasmin, kallikrein and pepsin but not for papain with synthetic and natural substrates. These inhibitors formed stable complexes with trypsin and chymotrypsin in an equimolar ratio, and their inhibitory mechanisms for both enzymes were of non-competitive type.     

Pancreatic proteolytic enzymes of ostrich purified on immobilized protein inhibitors. Characterization of a new form of chymotrypsin (Chtr1)

Four forms of chymotrypsin (Chtr1, Chtr2, Chtr3, Chtr4), one form of trypsin and one form of elastase were purified from a slightly alkaline extract of ostrich (Struthio camelus) pancreas. The zymogens in the crude extract were activated with immobilized trypsin and then separated by affinity chromatography using immobilized inhibitors and ion exchange chromatography. One of the purified forms of chymotrypsin (Chtr1) exhibited an unusual interaction with the highly selective protein trypsin inhibitor from Cucurbita maxima (CMTI). Interactions with other protein trypsin inhibitors such as basic pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (STI), trypsin inhibitors from Cyclanthera pedata (CyPTI), Cucurbita pepo (CPTI), Cucurbita pepo var. giramontia (CPGTI) and Linum usitatissimum (LUTI) were also investigated. This study demonstrated the affinity of Chtr1 to inhibitors containing Arg at P1 position. Studies of substrate specificity of Chtr1 using oxidized B-chain of insulin revealed four susceptible bonds: Tyr15-Leu16, Phe24-Phe25, Phe25-Tyr26 and, surprisingly, Arg22-Gly23. The amino acid composition, as well as the first 13 residues of the N-terminal amino acid sequence, was determined. Studies of ostrich elastase showed that it can interact with immobilized CMTI in the presence of 5 M NaCl. This unusual characteristic is reported for the first time and suggests that elastase specificity depends on ionic strength. The kinetic constants K(M), k(cat) and k(cat)/K(M) for purified ostrich trypsin, chymotrypsin 4 and elastase were also determined.     

Serine proteinase inhibitors from Vipera ammodytes venom. Isolation and kinetic studies

Three protein inhibitors of serine proteinases were isolated from the crude venom of the long-nosed viper Vipera ammodytes ammodytes by ion-exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki = 3.4 X 10(-10) and 5.6 X 10(-10) M), while the third one primarily inhibits chymotrypsin (Ki = 4.3 X 10(-9) M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N-terminal group in the former inhibitors is blocked; arginine is the N-terminal amino acid in the latter. Besides trypsin and alpha-chymotrypsin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.     

Low-molecular-weight trypsin inhibitors of microbial origin

Three hundred actinomyces cultures newly isolated from the soil of different regions of the Soviet Union were tested for their ability to produce inhibitors of trypsin-like proteases. Seven previously not known to produce trypsin inhibitors (Streptomyces bikiniensis 17-5, S. sporoclivatus 28-1, S. filamentosus 32-11, S. diastatochromogenes 20-4, S. lavendulae 29-4, S. violacens 52-8, and Streptoverticillium cinnamoneum 36-8) were found to possess high antitrypsin activity. The morphological and cultural properties of the strains and the dynamics of inhibitor production were investigated. S. bikiniensis 17-5 was studied in greatest detail. Its culture filtrate contained several inhibitors for trypsin and one for chymotrypsin. A mixture of oligopeptides with Mr of 300-500 was obtained by the described procedure which included the adsorption of the culture fluid filtrate on charcoal followed by ion-exchange chromatography on CM-cellulose. Four trypsin inhibitors (Sb-IT1, Sb-IT2, Sb-IT3, and Sb-IT4) were isolated from the mixture in a highly purified state by reversed-phase high-performance liquid chromatography. Sb-IT2 has been recognized as formylhistidylvaline with an Mr of 282. No trypsin inhibitor of this structure has been described previously.     

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants

Seven trypsin inhibitors were isolated from the seeds of Cucurbitaceae plants: two from cucumber (Cucumis sativus) and red bryony (Bryonia diotica) and one from figleaf gourd (Cucurbita ficifolia), spaghetti squash (Cucurbita pepo var. (vegetable spaghetti) and water melon (Citrullus vulgaris). The inhibitors were purified by fractionation with ammonium sulphate, followed by ion-exchange chromatography and affinity chromatography using immobilized trypsin or anhydro-trypsin. The homogeneous inhibitors from cucumber and water melon are made up of 32 and 30 amino acid residues, respectively, whereas the remaining ones of 29 residues. All inhibitors contain three disulphide bridges and are free of threonine, phenylalanine and tryptophan. Inhibitors from spaghetti squash and CSTI IIb from cucumber are inactivated by acetylation of free amino groups whereas the remaining ones are inactivated by modification of arginine with 1,2-cyclohexanedione. Thus the P1 residues of the reactive sites of the inhibitors are lysine and arginine, respectively.     

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.     

Isolation and characterization of the trypsin inhibitors from winged bean seed (Psophocarpus tetragonolobus (L) Dc.).

The trypsin inhibitors from winged bean seed were isolated by affinity chromatography on trypsin-Sepharose 4B and the components fractionated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The major components, inhibitors 2 and 3 were found to be homogeneous proteins with molecular weights of about 20,000. The inhibitors stoichiometrically inhibited bovine trypsin in the molar ratio of 1 : 1 whereas the inhibition of bovine alpha-chymotrypsin was weak and non-stoichiometric. Amino acid analysis indicated that both the inhibitors contain four cysteine residues and are rich in aspartic acid, glutamic acid, glycine, valine and leucine; however, inhibitor 3 lacks histidine and methionine while inhibitor 2 contains one histidine and three methionines. A minor trypsin inhibitor fraction was also isolated which contained at least three proteins with a molecular weight of about 10,000 and a high content of half-cystine.     

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.     

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.     

Isolation and properties of navy bean proteinase inhibitor component I.

A family of protease inhibitors (of which one was shown to be homogeneous and one nearly so by ion-exchange chromatography, gel filtration, disc electrophoresis, and amino-terminal analysis) was obtained from navy beans. Amino acid analysis demonstrated the inhibitors to be similar in composition and size to the Bowman-Birk and other low-molecular-weight inhibitors from plants. Interaction with trypsin was monitored by examining enzyme-inhibitor complex formation and inhibitor modification in acrylamide gels after pH 8 and pH 4 preincubation with varying concentrations of trypsin. Specific inhibiting capacities, determined with seven substrates after preincubation at the above pH values, varied greatly with different substrates and were less after low pH preincubation.     

Isolation of soybean trypsin inhibitors by affinity chromatography on anhydrotrypsin-Sepharose 4B

By repeated treatments of trypsin with phenylmethylsulfonyl fluoride (PMSF), followed by base elimination of PMS from the PMS-trypsin, a catalytically inactive anhydrotrypsin preparation of low (less than 1%) active trypsin content was obtained. Inactive material was removed by affinity chromatography on trypsin inhibitor-Sepharose 4B and the purified anhydrotrypsin with full binding capacity for trypsin inhibitors was coupled to cyanogen bromide-activated Sepharose 4B. When used below its maximum capacity for trypsin inhibitors the anhydrotrypsin-Sepharose-4B affinity column absorbed both classes of inhibitors present in soybean. When overloaded, the Kunitz type was bound preferentially. Based on this observation, conditions for the partial separation of the two types of inhibitors were worked out.     

Multiple trypsin inhibitors from Momordica cochinchinensis seeds, the Chinese drug mubiezhi

Five trypsin inhibitors, with N-terminal sequences demonstrating homology to each other and exhibiting a molecular weight of 5100, 4800, 4400, 4100, and 3900, respectively, were isolated from Momordica cochinchinensis seeds with a protocol involving acid extraction, ion exchange chromatography on SP-Sepharose chromatography, and RP-HPLC on a C18 column. Specific inhibitory activity against trypsin was demonstrated by the trypsin isoinhibitors with Ki values ranging from 5.3 x 10(-8) to 1.8 x 10(-6) M. None of the isoinhibitors could be cleaved by trypsin.     

Kunitz-type inhibitors in human serum. Identification and characterization

Human serum contains small amounts (approximately 0.1 mg/liter) of two protein protease inhibitors of low molecular weight (approximately 6500) and basic isoelectric point (Kunitz-type). They were purified by affinity chromatography on immobilized trypsin and ion-exchange chromatography in the fast protein liquid chromatography system. Their chemical, immunochemical, and functional properties indicate that the purified inhibitors are highly homologous with the basic pancreatic trypsin inhibitor which is widely distributed in bovids and caprids. Their inhibitory activity toward serine proteases such as plasmin and kallikrein suggests a possible regulatory role in blood clotting and fibrinolysis.     

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).     

Formation of Bowman-Birk inhibitors during the germination of horsegram (Dolichos biflorus)

Three Bowman-Birk type inhibitors (HGGI-I, II and III), which appear in the cotyledons of 120 h germinated horsegram (Dolichos biflorus) seeds have been purified to homogeneity by size-exclusion chromatography and ion-exchange chromatography. HGGI-I, HGGI-II and HGGI-III differ from each other and from the dormant seed inhibitors in amino acid composition, molecular size and charge. The amino-terminal sequence analyses indicate that these inhibitors are derived from the isoinhibitors of the dormant seed by a limited proteolysis and not by de novo synthesis. These inhibitors differ from each other at their amino-terminus. HGGI-II identical to HGGI-I except for the loss of a single amino-terminal aspartyl residue, where as HGGI-III shows the loss of a pentapeptide. All the three inhibitors are potent competitive inhibitors of trypsin and chymotrypsin. The dissociation constants (K(i)s) for trypsin inhibition indicate that amino-terminal tail of the inhibitors play a role in trypsin binding probably through electrostatic interaction.     

苦荞胰蛋白酶抑制剂的纯化及特性研究

利用固相胰蛋白酶亲和色谱及离子交换色谱,从苦荞种子中分离纯化了三个具有抑制胰蛋白酶活力的组份(BTI-Ⅰ,Ⅱ,Ⅲ)。经聚丙稀酰胺凝胶等电聚焦测定,三种抑制剂的等电点为6.87,6.7,5.14。根据Sephadex G-75凝胶过滤,SDS-PAGE测定,以及由当量抑制比值和氨基酸组份的推算,它们的分子量范围分别为5200-5700(Ⅰ),5500—6000(Ⅱ),5000—5600(III)。三者对胰蛋白酶的抑制常数分别为2.12×10~(-8),4.78×10~(-9),1.22×10~(-8)。氨基酸分析结果表明,它们均含有很高的极性氨基酸。在酸性条件下,它们均具有很高的热稳定性。化学修饰的结果表明,它们活性中心的氨基酸残基为Lys。     

Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds

Seven new trypsin inhibitors, CyPTI I-VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28-30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine beta-trypsin (K(a)>10(11) M(-1)) and, more weakly, bovine alpha-chymotrypsin (K(a) approximately 10(4)-10(6) M(-1)). In the presence of 3 M NaCl the association constants of CyPTIs with alpha-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25-Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Double-headed protease inhibitors from black-eyed peas. I. Purification of two new protease inhibitors and the endogenous protease by affinity chromatography.

Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3 M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.