Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea.

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ±â€¯4.5 nM) and chymotrypsin (Ki = 807.8 ±â€¯23.7 nM) while RsKI (Ki = 172.0 ±â€¯9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > ß-sheets/ß-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.

Protein Separation Coacervation with Carboxymethyl Cellulose of Different Substitution Degree: Noninteracting Behavior of Bowman-Birk Chymotrypsin Inhibitor.

We first observed that protein/polysaccharide interaction exhibited noninteracting behavior which makes Bowman-Birk chymotrypsin inhibitor (BBI) always free of complexation, being separated from another protein with similar isoelectric points, Kunitz trypsin inhibitor (KTI). Turbidity titrations showed that the electrostatic attractions were much stronger between KTI/BBI (KBi) and carboxymethyl cellulose of higher substitution degree. Unchanged chymotrypsin inhibitory activity (CIA) indicated that BBI had negligible contribution to protein recovery and trypsin inhibitory activity (TIA). Tricine-SDS-PAGE revealed that, at r = 20:1-2:1, unbound BBI was left in the supernatant when bound KTI transferred into precipitates, even if there was excess negative charge. Thus, purified KTI or BBI was achieved easily at the given conditions. The noninteracting behavior of BBI was further confirmed by ITC, where the binding enthalpy of BBI to CMC was negligible compared with the high binding affinity ( Kb) of KTI. This work will be beneficial to protein purification based on protein-polysaccharide coacervation.

Purification of lectin and Kunitz trypsin inhibitor from soya seeds.

The search for potent and selective therapeutic agents is progressing by the study of natural compounds in plants. Plant-derived macromolecules are considered emerging therapeutic agents and an alternative to synthetic and small molecule drugs. Where it has long been known that plants possess medicinal properties, the compounds responsible for their action are in many cases still unknown: often only whole crude plant extracts or fractionated extracts are tested for the ability to inhibit common pathogens. Here, we present a fast protein liquid chromatography method for the separation of crude plant proteins. Kunitz trypsin inhibitor (KTI; 24.2 kDa) and lectin (31 kDa) were purified from Glycine max by liquid extraction followed by ion exchange column chromatography. The need for serial chromatographic separation steps has been eliminated by introducing more complex elution profiles hence reducing cost, time and improving recovery. The identity of KTI-A and lectin was confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF MS). Cell proliferation assays using B16F1 melanoma cells revealed that both KTI and the monomeric lectin retained some antiproliferative activity. This method could be useful for rapid and cost-effective purification of bioactive compounds from plant material.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity.

Allelic differentiation of Kunitz trypsin inhibitor in wild soybean (Glycine soja).

Soybean Kunitz trypsin inhibitor (SKTI) has several polymorphic types, which are controlled by co-dominant multiple alleles at a single locus. Of these types, Tia and Tib are predominant types, and there are nine differences in amino acids between Tia and Tib. Recently, an intermediate transitional type (Tibi5) between them was detected. However, other transitional types have not been detected despite surveys of many cultivated and wild soybeans. One of the reasons why other transitional variants have not been found is inferred to be due to the difficulty of the detection of SKTI protein variants by polyacrylamide gel electrophoresis (PAGE). To detect novel variants of SKTI, nucleotide sequence analysis in addition to PAGE was carried out. Four new variants were found from many Japanese wild soybeans. Of these variants, three (designated as Tiaa1, Tiaa2, Tiab1) were detected through gene sequence analysis on wild soybeans having the same electrophoretic mobility as Tia, and one (Tig) was detected through PAGE. The Tig variant showed a slightly lower electrophoretic mobility than Tic. The nucleotide sequences of Tig were identical to those of Tib except for one T-->C transitional mutation at position +340. The sequences of Tiaa1 and Tiaa2 genes were identical to those of Tia with the exception of a G-->A mutation at position +376 and a T-->C mutation at +404, respectively. The sequence of Tiab1 differed from Tia by three nucleotides: C-->A at position +331, T-->C at +459 and A-->G at +484. Of the three nucleotide changes, two were common to Tiab1, Tibi5 and Tib, suggesting that Tiab1 is an intermediate transitional type between Tia and Tib. Our results suggest that Tib type has been differentiated through a series of mutations from Tia before the domestication of cultivated soybean.

Proteomic characterization of inter-alpha inhibitor proteins from human plasma.

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.

A kunitz-type protease inhibitor bikunin disrupts ligand-induced oligomerization of receptors for transforming growth factor (TGF)-beta and subsequently suppresses TGF-beta signalings.

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses transforming growth factor-beta1 (TGF-beta1)-stimulated expression of urokinase-type plasminogen activator (uPA) in human ovarian cancer cells that lack endogenous bik. In the present study, we tried to elucidate the mechanism by which bik also inhibits plasminogen activator inhibitor type-1 (PAI-1) and collagen synthesis using human ovarian cancer cells. Here, we show that (a) there was an enhanced production of both uPA and PAI-1 in HRA cells in response to TGF-beta1; (b) the overexpression of bik in the cells or exogenous bik results in the inhibition of TGF-beta1 signaling as measured by phosphorylation of the downstream signaling effector Smad2, nuclear translocation of Smad3, and production of PAI-1 and collagen; (c) bik neither decreased expression of TGF-beta receptors (TbetaRI and TbetaRII) in either cell types nor altered the specific binding of 125I TGF-beta1 to the cells, indicating that the effects of bik in these cells are not mediated by ligand sequestration; (d) TbetaRI and TbetaRII present on the same cells exclusively form aggregates in TGF-beta1-stimulated cells; (e) co-treatment of TGF-beta1-stimulated cells with bik suppresses TGF-beta1-induced complex formation of TbetaRI and TbetaRII; and (f) a chondroitin-4-sulfate side chain-deleted bik (deglycosylated bik) does not inhibit TGF-beta1 signaling or association of type I/type II receptor. We conclude that glycosylated bik attenuates TGF-beta1-elicited signaling cascades in cells possibly by abrogating the coupling between TbetaRI and TbetaRII and that this probably provides the mechanism for the suppression of uPA and PAI-1 expression.

Cross-inhibitory activity of cereal protein inhibitors against alpha-amylases and xylanases.

The purification and characterisation of a xylanase inhibitor (XIP-I) from wheat was reported previously. In our current work, XIP-I is also demonstrated to have the capacity to inhibit the two barley alpha-amylase isozymes (AMY1 and AMY2). XIP-I completely inhibited the activity of AMY1 and AMY2 towards insoluble Blue Starch and a soluble hepta-oligosaccharide derivative. A ternary complex was formed between insoluble starch, a catalytically inactive mutant of AMY1 (D180A), and XIP-I, suggesting that the substrate-XIP-I interaction is necessary for inhibition of barley alpha-amylases. K(i) values for alpha-amylase inhibition, however, could not be calculated due to the nonlinear nature of the inhibition pattern. Furthermore, surface plasmon resonance and gel electrophoresis did not indicate interaction between XIP-I and the alpha-amylases. The inhibition was abolished by CaCl(2), indicating that the driving force for the interaction is different from that of complexation between the barley alpha-amylase/subtilisin inhibitor (BASI) and AMY2. This is the first report of a proteinaceous inhibitor of AMY1. BASI, in addition, was demonstrated to partially inhibit the endo-1,4-beta-D-xylanase from Aspergillus niger (XylA) of glycoside hydrolase family 11. Taken together, the data demonstrate for the first time the dual target enzyme specificity of BASI and XIP-I inhibitors for xylanase and alpha-amylase.

One-step purification of Kunitz soybean trypsin inhibitor.

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.

Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli.

Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.

In acute inflammation, the chondroitin-4 sulphate carried by bikunin is not only longer, it is also undersulphated.

Bikunin (Bk) is a Künitz-type serine proteinase inhibitor, which occurs in human plasma, mainly as covalent complexes with one or two of the three peptide heavy chains. The leading member of this glycoprotein family is inter-alpha-inhibitor (I alpha I), which consists of two heavy chains (H1 and H2) linked to Bk. Bk carries a glycosaminoglycan (GAG) chain, which is linked by ester bonds to the heavy chains of I alpha I. Furthermore, Bk, I alpha I and related components such as pre-alpha-inhibitor (P alpha I), all together making up the I alpha I family, present antiinflammatory and antimetastatic effects that hinge on this GAG chain. Recently (Eur. J. Biochem. 268 (2001) 2717), we provided evidence that, during acute phase response, the GAG chain of Bk, which is a low-sulphated chondroitin-sulphate, increases in size according to the severity of the inflammatory disease. This increase affects Bk-containing proteins in circulating blood as well as Bk excreted in higher amounts in urine of these patients. In this work, we have more extensively analysed the GAG chain of Bk isolated from urine collected from a unique patient with septic shock. Using MALDI-TOF-MS and HPLC analyses of chondrodisaccharides released by enzymatic digestion, we have demonstrated that the GAG chain is clearly modified; it consists of 20 +/- 5 disaccharide units vs. 14 +/- 3 for reference Bk originating from healthy donors. Among them, only 3 +/- 2.5 units are 4-sulphated for patient's Bk vs. 5 +/- 1.5 for reference Bk. Therefore, the non-sulphated region of the GAG chain, which is located towards its non-reducing end, where the heavy chains are positioned, is lengthened from 9 for reference Bk to 17 disaccharide units. We suggest that the biological effects of Bk-proteins may hereby be modulated during inflammatory diseases.

Characterization of a Kunitz trypsin inhibitor with one disulfide bridge purified from Swartzia pickellii.

Swartzia pickellii is a Leguminosae that belongs to the Caesalpinioideae sub-family the Swartzia pickellii Trypsin Inhibitor (SWTI), a serine proteinase inhibitor was isolated from its seeds. SWTI is a single polypeptide chain protein and it's structure has 174 amino acid residues, it homologous to other Kunitz plant inhibitors, however shows some major differences: it contains only one disulfide bridge, instead two which are usually found in plant Kunitz inhibitors, and the SWTI reactive site does not contain the usual Arg or Lys residues at the putative reactive site (position 65). A glycosylation site was detected at Asn38 with 1188 kDa carbohydrate portion. The primary structure micro heterogeneity was found combining the sequence determination and mass spectrometry. Three forms of SWTI were actually defined: two glycosylated forms a 20,204 kDa (Arg 165) and 20,185 kDa (His 165) and one deglycosylated form 19,016 kDa (Arg 165), all of them contain a Met residue at position 130.

Three-phase affinity partitioning of proteins.

Three-phase partitioning is an elegant way to separate proteins directly from even the large volumes of crude suspensions. It was found that interfacing it with a metal-affinity-based step makes the technique highly selective. As "proof of the concept," soybean trypsin inhibitor was purified 13-fold with 72% recovery. As recombinant proteins (via their polyhistidine tags) and many other naturally occurring proteins are often purified by immobilized metal ion affinity chromatography, the technique described here should prove valuable in purifying biotechnologically important proteins at a large scale.

Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate-proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester.

A gel electrophoresis apparatus capable of scanning the migration path fluorometrically and of computer-directed electroelution of bands was applied to the mass spectrometric identification of sequentially electroeluted 5(6)-carboxyfluorescein-N-hydrosuccinimide ester (FLUOS)-labeled sodium dodedyl sulfate (SDS)-proteins. The masses of four electroeluted SDS-proteins under study determined by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry are changed by 1% due to their reaction with FLUOS in a 1:5 molar ratio of protein:label, allowing for the identification of the labeled intact proteins on the basis of mass. More importantly, the partial (10 or 50%) derivatization of proteins with FLUOS does not preclude their tryptic hydrolysis, and identification of the protein on the basis of the mass spectrometric analysis of its tryptic peptides. Potentially, the procedure allows for the automated mass spectrometric identification of SDS-proteins globally labeled with FLUOS and electrophoretically separated, without need for any gel sectioning.

Selectivity of IMAC columns in trypsin inhibitor purification.

The properties of an adsorbent and the parameters in an adsorption process affect the resolution of chromatographic purifications. This is reflected in the elution profile, which shows the relative affinity of different proteins for a specific adsorbent. In the work presented here, elution profiles for trypsin inhibitor were used to study the effects of the concentration of trypsin inhibitor, ionic strength of the protein solution, slope of the elution gradient, and the regeneration treatment of the chromatography column on the selectivity of the adsorbent Cellufine Chelate-Cu(II)(ida). Cytochrome c was used as a reference protein. Variations in the concentrations of trypsin inhibitor and in the ionic strength of the buffered solution did not have any effects on the elution profile. On the other hand, changes in the slope of the pH gradient used for elution caused shifting of the elution peaks toward lower values of the elution volume, resulting in the best strategy to modify the elution profile of the system. Finally, using a constant slope pH gradient of elution, the variation of the selectivity of the adsorbent for trypsin inhibitor when subjected to cleaning treatments with 0.5 N NaOH was studied. Appropriate cleaning practices used in industry were followed. The adsorbent showed only a slight tendency for resolution loss in the order of 2 x 10(-4) days(-1). The results presented here show a good stability of the adsorbent when compared to other biospecific adsorbents commonly used.

Optimal synthesis of protein purification processes.

There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high-resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches.

Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

Purification, crystallization and preliminary crystallographic study of a Kunitz-type trypsin inhibitor from Delonix regia seeds.

The Kunitz-type trypsin inhibitor from seeds of Flamboyant (Delonix regia) has been purified to homogeneity and plate-like crystals suitable for X-ray analysis have been grown by the hanging-drop method using PEG 6000 as a precipitant. The crystals belong to space group P2(1)2(1)2(1) with unit-cell parameters a = 32.15, b = 69.39, c = 72.54 A. X-ray diffraction data have been collected to 2.95 A resolution. The structure has been solved by molecular replacement using the known structures of trypsin inhibitors from Erythrina caffra seeds (PDB code 1tie) and from soya beans (Glycine max; PDB code 1ba7) as search models.

[New studies on natural inhibitors of proteolytic enzymes]. / Novoe o prirodnykh ingibitorakh proteoliticheskikh fermentov.

New data on proteolytic enzyme inhibitors and mechanisms of their interaction with the enzymes are reviewed. In recent years, a number of new inhibitors comprising families earlier unknown have been described such as proteins from the parasitic nematode Ascaris lumbricoides, ecotin from the periplasm of Escherichia coli, proteins PMP-C and PMP-D from locust Locusta migratoria, and hirustasin from the medicinal leech Hirudo medicinalis. At the same time, some proteins that may be assigned to inhibitors on the basis of their structures were found to perform other (not inhibitory) functions. Thus, the family of the Kunitz soybean trypsin inhibitor includes plant storage proteins and proteins whose synthesis is induced by stress factors. Numerous inhibitors interacting with the enzymes by mechanisms other than the substrate-like ones were identified, such as ornithodorin and anticoagulant peptide from tick Ornithodoros moubata (inhibitors of the blood clotting system proteases), an inhibitor from snake (Bothrops jararaca) venom, and ecotin, an inhibitor of serine proteases with an unusually broad specificity range. Special emphasis is placed on enzyme inhibition with propeptides and the mechanism of this process.

Potato tuber protein proteinase inhibitors belonging to the Kunitz soybean inhibitor family.

Three protein inhibitors of proteolytic enzymes with molecular weights 21, 22, and 23 kD were isolated from potato tubers (Solanum tuberosum L.) by ammonium sulfate precipitation followed by gel and ion-exchange chromatography. The 21- and 22-kD proteins were shown to be serine proteinase inhibitors with different specificities. The 21-kD protein inhibits human leucocyte elastase and trypsin effectively, but it is less effective towards chymotrypsin. The 22-kD protein is an inhibitor of cysteine proteinases and suppresses the activities of papain, ficin, and bromelain with the same affinities. None of the isolated proteins inhibit subtilisin, pepsin, or cathepsin D. The 21-kD protein consists of two disulfide-linked polypeptide chains with molecular weights of 16.5 +/- 1 kD and 4.5 +/- 1 kD. The 22-kD and 23-kD proteins have a single polypeptide chain. The N-terminal 22-25 amino acid sequences of these three proteins were determined. These sequences have significant homology to other plant inhibitors from the Kunitz soybean inhibitor superfamily.