Structure and stability of the potato cysteine protease inhibitor group (cv. Elkana)

The conformational stability of potato cysteine protease inhibitor (PCPI), the second most abundant protease inhibitor group in potato tuber, was investigated at ambient temperature and upon heating using far- and near-UV circular dichroism spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC). The PCPI isoforms investigated have a highly similar structure at both the secondary and the tertiary level. PCPI isoforms show structural properties similar to those of the potato serine protease inhibitor group and the Kunitz type soybean trypsin inhibitor, a known beta-II protein. Therefore, PCPI isoforms are also classified as members of the beta-II protein subclass. Results show that the thermal unfolding of PCPI isoforms does not follow a two-state mechanism and that at least one intermediate is present. The occurrence of this intermediate is most apparent in the thermal unfolding of PCPI 8.3 as indicated by the presence of two peaks in the DSC thermogram. Additionally, the formation of aggregates (>100 kDa), especially at low scan rates, increases the apparent cooperativity of the unfolding.     

The most abundant protease inhibitor in potato tuber (cv. Elkana) is a serine protease inhibitor from the Kunitz family

The gene of the most abundant protease inhibitor in potato cv. Elkana was isolated and sequenced. The deduced amino acid sequence of this gene showed 98% identity with potato serine protease inhibitor (PSPI), a member of the Kunitz family. Therefore, the most abundant protease inhibitor was considered to be one of the isoforms of PSPI. The PSPI group represents approximately 22% of the total amount of proteins in potato cv. Elkana and is composed of seven different isoforms that slightly differ in isoelectric point. Antibodies were raised against the two most abundant isoforms of PSPI. The binding of these antibodies to PSPI isoforms and protease inhibitors from different groups of protease inhibitor in potato showed that approximately 70% of the protease inhibitors present in potato juice belong to the Kunitz family.     

Molecular properties and activities of tuber proteins from starch potato cv. Kuras

Potato starch production leaves behind a huge amount of juice. This juice is rich in protein, which might be exploited for food, biotechnological, and pharmaceutical applications. In northern Europe cv. Kuras is dominant for industrial starch production, and juice protein of freshly harvested mature tubers was fractionated by Superdex 200 gel filtration. The fractions were subjected to selected activity assays (patatin, peroxidase, glyoxalases I and II, alpha-mannosidase, inhibition of trypsin, Fusarium protease, and alcalase) and protein subunit size determination by SDS-PAGE and mass spectrometry. Proteins present in SDS-PAGE bands were identified by tryptic peptide mass fingerprinting. Protein complexes such as ribosomes and proteasomes eluted with the void volume of the gel filtration. Large proteins were enzymes of starch synthesis dominated by starch phosphorylase L-1 (ca. 4% of total protein). Five identified dimeric patatin variants (25%) coeluted with four monomeric lipoxygenase variants (10%) at 97 kDa. Protease inhibitor I variants (4%) at 46 kDa (hexamer) inhibited alcalase. Fourteen Kunitz protease inhibitor variants (30%) at 19 kDa inhibited trypsin and Fusarium protease. Carboxypeptidase inhibitor variants (5%) and defensins (5%) coeluted with phenolics. The native sizes and molecular properties were determined for 43 different potato tuber proteins, several for the first time.     

Immunoassays of soy proteins

Proteins of soybeans (Glycine max) are widely used in animal and human nutrition. In addition to the bulk of the seed storage proteins, which are classified as albumins and globulins, approximately 6% of soybean proteins are classified as inhibitors of trypsin and chymotrypsin and approximately 0.5% are sugar-binding lectins. The two major classes of inhibitors are the Kunitz trypsin inhibitor, which inhibits trypsin, and the Bowman-Birk inhibitor (BBI), which inhibits both trypsin and chymotrypsin. Unless removed or inactivated, these inhibitors and lectins can impair the nutritional quality and safety of soy-based diets. On the other hand, several studies suggest that BBI can also function as an anticarcinogen, possibly through interaction with a cellular serine protease. Good-quality soybean proteins contribute to the nutritional value of many specialty foods including infant soy formulas and milk replacers for calves, and provide texture to many processed foods. However, they may also induce occasional allergic responses in humans. This paper outlines immunoassays developed to analyze for soy proteins in different soybean lines, in processed foods, and in nonsoy foods fortified with soy proteins. An assessment of the current status of immunoassays, especially of enzyme-linked immunosorbent assays for soybean inhibitors of digestive enzymes, soy globulins, and soy lectins, demonstrates the usefulness of these methods in plant and food sciences and in medicine.     

Kunitz-type serine protease inhibitor from potato (Solanum tuberosum L. cv. Jopung)

An antifungal protein, AFP-J, was purified from tubers of the potato (Solanum tuberosum cv. L Jopung) by various chromatographic columns. AFP-J strongly inhibited yeast fungal strains, including Candida albicans, Trichosporon beigelii, and Saccharomyces cerevisiae, whereas it exhibited no activity against crop fungal pathogens. Automated Edman degradation determined the partial N-terminal sequence of AFP-J to be NH2-Leu-Pro-Ser-Asp-Ala-Thr-Leu-Val-Leu-Asp-Gln-Thr-Gly-Lys-G lu-Leu-Asp-Ala-Arg-Leu-. The partially sequence had 83% homology with a serine protease inhibitor belonging to the Kunitz family, and the protein inhibited chymotrypsin, pepsin, and trypsin. Mass spectrometry showed that its molecular mass was 13 500.5 Da. This protease inhibitor suppressed over 50% the proteolytic activity at 400 microg/mL. These results suggest that AFP-J is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

Structural characterization of potato protease inhibitor I (Cv. Bintje) after expression in Pichia pastoris

In the present study the structural properties of potato protease inhibitor 1 (PI-1) were studied as a function of temperature to elucidate its precipitation mechanism upon heating. A cDNA coding for PI-1 from cv. Bintje was cloned and expressed in Pichia pastoris. Using the recombinant PI-1 it was suggested that PI-1 behaves as a hexameric protein rather than as a pentamer, as previously proposed in the literature. The recombinant protein seems either to have a predominantly unordered structure or to belong to the beta-II proteins. Differential scanning calorimetry analysis of PI-1 revealed that its thermal unfolding occurs via one endothermic transition in which the hexameric PI-1 probably unfolds, having a dimer instead of a monomer as cooperative unit. The transition temperature for the recombinant PI-1 was 88 degrees C. Similar results were obtained for a partially purified pool of native PI-1 from cv. Bintje.     

Kinetics of the inhibition of fusarium serine proteinases by barley (Hordeum vulgare L.) inhibitors

Fungal infections of barley and wheat cause devastating losses of these food crops. The endogenous proteinase inhibitors produced by plant seeds probably defend the plants from pathogens by inhibiting the degradation of their proteins by the pathogen proteases. We have studied the interactions of barley grain inhibitors with the subtilisin-like and trypsinlike proteinases of Fusarium culmorum. The inhibition kinetics of three inhibitor proteins, chymotrypsin/subtilisin inhibitor 2 (CI-2), barley alpha-amylase/subtilisin inhibitor (BASI), and Bowman-Birk trypsin inhibitor (BBBI), have been studied in detail for the first time using fungal enzymes. The kinetic studies were performed at physiological pH values to mimic in vivo conditions. Numerical approaches to kinetic analyses were used to calculate the inhibition constants, because the data analyses were complicated by some inhibitor turnover and the instability of enzymes and substrates. All were slow, tight-binding inhibitors that followed either a two-step mechanism (CI-2 and BASI) or a single-step mechanism (BBBI) under the conditions investigated. The overall Ki values derived were approximately 50 pM, 1 nM, and 0.1 nM for CI-2, BASI, and BBBI, respectively. The main difference between the CI-2 and the BASI inhibitions was accounted for by the stabilities of their final complexes and the rate constants for their second dissociation steps (9 x 10(-6)/s and 3 x 10(-4)/s, respectively). Understanding the inhibition mechanisms will be valuable in designing improved strategies for increasing the resistance of the grains to fungal infections.     

Abundant class III acidic chitinase homologue in tamarind (Tamarindus indica) seed serves as the major storage protein

The phyla Leguminosae contains protease inhibitors, lectins, chitinases, and glycohydrolases as major defense proteins in their seeds. Electrophoretic analysis of the seed proteins of tamarind ( Tamarindus indica L.), an agri-waste material, indicated the unusual presence of two major proteins comparable to overexpression of recombinant proteins. These proteins were identified by amino-terminal analysis to be (1) Kunitz-type trypsin inhibitor and (2) class III endochitinase (34000 Da). These two proteins were purified to apparent homogeneity by a single-step chitin bead affinity chromatography and characterized. The Kunitz inhibitor was specific toward inhibiting trypsin with a stoichiometry of 1:1. The 33000 +/- 1000 Da protein, accounting for >50% of the total seed protein, is an acidic glycoprotein exhibiting a very low endotype hydrolytic activity toward chitin derivatives. SDS-PAGE followed by densitometry of tamarind seed germination indicates the disappearance of the chitinase with the concomitant appearance of a cysteine endopeptidase. On the basis of its abundance, accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity, this 34000 Da protein designated "tamarinin" physiologically serves as the major storage protein.     

Determination of Pea Proteinase Inhibitors Using ELISA Based on Monoclonal Antibodies

Abstract

An immunoassay method for analysis of trypsin and chymotrypsin inhibitor activity in seeds from different pea cultivars has been developed. This method is fast, cheap and well suited for screening of large numbers of seed samples. The technique is based on enzyme linked immunoassay (ELISA) using monoclonal antibodies produced against pea (Pisum sativum L.) proteintype trypsin inhibitors (PPI). The results obtained by ELISA have been compared with results achieved by using traditional enzymatic analyses for determination of both trypsin and chymotrypsin inhibitor contents, and these methods showed good agreement. Great differences in PPI levels have been found between various pea cultivars grown in Finland and Denmark, and these differences comprised both the total amount of PPI and the relative composition of individual PPI. Several proteins occurring in pea exhibited inhibitor activity, and at least 10 PPI inhibitors with p/ values 4.9–7.8 were detected. Pea cultivars with low PPI levels had a PPI composition different from the PPI composition found in cultivars with high PPI levels.     

Pea (Pisum sativum L.) protease inhibitors from the Bowman-Birk class influence the growth of human colorectal adenocarcinoma HT29 cells in vitro

The Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybean has been described as a potential cancer chemopreventive agent. We have compared the effects of BBI with those of two variant recombinant pea (Pisum sativum L.) seed protease inhibitors, rTI1B and rTI2B, homologous to BBI but differing in inhibitory activity, on the growth of human colorectal adenocarcinoma HT29 cells in vitro. A significant and dose-dependent decrease in the growth of HT29 cells was observed using all protease inhibitors, with rTI1B showing the largest decrease (IC50 = 46 microM). Inclusion of the pan-caspase inhibitor, Boc-D-FMK, did not negate the effects of rTI1B or rTI2B in the cell assays. The relative effectiveness of rTI1B and rTI2B may correlate with a variant amino acid sequence within their respective chymotrypsin inhibitory domain, in agreement with a chymotrypsin-like protease as a potential target.     

Complete amino acid sequence of the lentil trypsin-chymotrypsin inhibitor LCI-1.7 and a discussion of atypical binding sites of Bowman-Birk inhibitors

The complete primary structure of the lentil (Lens culinaris) trypsin-chymotrypsin inhibitor LCI-1.7 was determined by conventional methods in order to find relationships between partial sequences and the difference in action against human and bovine chymotrypsin. As other Bowman-Birk type inhibitors, LCI-1.7 contained 68 amino acid residues, seven disulfide bridges, and two reactive sites, Arg16-Ser17 for trypsin and Tyr42-Ser43 for chymotrypsin. Evaluation of sequence homologies showed that it belonged to the group III Bowman-Birk inhibitors. The atypical additional binding site of LCI-1.7 for human chymotrypsin was discussed and compared with such binding sites of two other Bowman-Birk inhibitors, the Bowman-Birk soybean proteinase inhibitor BBI, and the lima bean proteinase inhibitor LBI I, for human and bovine trypsin and chymotrypsin. A concept to reduce the action of these inhibitors against human enzymes by genetic engineering was proposed.     

Improving digestibility of soy flour by reducing disulfide bonds with thioredoxin

The Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI) of trypsin and chymotrypsin contain disulfide bonds. Glycinin, the major storage protein in soybeans also contains disulfide bonds. Treatment of soy white flour with a NADP-thioredoxin system (NTS) effectively reduced disulfide bonds in soy flour and increased protein digestibility by trypsin and pancreatin as measured by the pH stat method. Treatment of soy flour with NTS increased the digestibility compared to soy white flour by 29.3 and 60.6% for trypsin and pancreatin, respectively. NTS-treated soy flour had similar digestibility by trypsin to autoclaved soy flour and casein, but digestibility by pancreatin was less than autoclaved soy flour and casein. The degree of reduction by NTS was highly correlated to the degree of hydrolysis (DH) by trypsin (R(2) = 0.93) and pancreatin (R(2) = 0.99). The DH of NTS-treated soy flour by trypsin is reflective of both inactivation of trypsin inhibitors and overall protein digestibility while pancreatin hydrolysis is reflective of only overall protein digestibility.     

Effects of pH and heat treatments on the structure and solubility of potato proteins in different preparations

The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.     

Isolation and properties of a Kunitz-type protein inhibitor obtained from Pithecellobium dulce seeds

We report for the first time the isolation and characterization of a protease inhibitor from the seeds of Pithecellobium dulce, which is a Leguminosae tree native to Mexico. The purification of the P. dulce trypsin inhibitor (PDTI) was a direct process. After its extraction (pH 8.0) and precipitation (80% (NH(4))(2)SO(4)), the pH was adjusted to 4.0, the supernatant was loaded onto a CM-Sepharose column, and a single peak of trypsin inhibitory activity was eluted (CM-TIA). The main component of CM-TIA was PDTI, a protein composed of two polypeptide chains joined by disulfide bridge(s), with a pI of 4.95 and a molecular weight determined by electrospray mass spectrometry of 19 614 Da. The N-terminal sequence of PDTI has the highest similarity with the seed inhibitor of Acacia confusa. PDTI lacks chymotrypsin inhibitory activity. A low rate of cytotoxicity of CM-TIA toward RINm5F cells contrasted with a high rate of the active fraction G75-TIA (gel filtration chromatography; LC(50) of 0.04 mg/mL).     

Sulfur compounds reduce potato toxins during extrusion cooking.

Free sulfhydryl groups in sulfur compounds have been reported to act directly on natural toxins to reduce toxicity. The objective of this study was to reduce protease inhibitors and glycoalkaloids in simulated snack foods by the addition of sulfur-containing compounds prior to extrusion. Thiamine, methionine, and benzyl disulfide were added to potato flakes at levels of 0.5% or 1.0% prior to twin-screw extrusion. Total and free thiols and protease inhibitors were monitored before and after extrusion by colorimetric assays. Potato glycoalkaloids were analyzed by HPLC and by immunoassay. Extrusion reduced potato flake disulfide bonds; disulfide bonds were higher in samples containing added sulfur compounds. Trypsin inhibitor activity was reduced by as much as 79% by extrusion plus methionine. Extrusion significantly reduced carboxypeptidase inhibitor, but only when benzyl disulfide and 0.5% methionine were not added. One percent methionine and thiamine resulted in 60% reductions in glycoalkaloids.     

Inhibition of Angiotensin converting enzyme I caused by autolysis of potato proteins by enzymatic activities confined to different parts of the potato tuber

Autolysis of protein isolates from vascular bundle and inner tuber tissues of potato (Solanum tuberosum) enhanced the inhibition of the angiotensin converting enzyme I (ACE), a biochemical factor affecting blood pressure (hypertension). The physiological age of the tuber affected the strength of ACE inhibition, the rate of its increase during autolysis, and the tuber tissue where ACE inhibition was most pronounced. The highest inhibitory activities (50% reduction in ACE activity achieved following autolysis at a protein concentration of 0.36 mg mL (-1)) were measured in tubers after 5-6 months of storage prior to sprouting. The rate of ACE inhibition was positively correlated with protease activity in tuber tissues. Amendment of the autolysis reaction with protein substrates from which bioactive ACE-inhibitory peptides may be released, for example, a purified recombinant protein or a concentrate of total tuber proteins, also enhanced ACE inhibition. Many tuber proteins including aspartic protease inhibitors were degraded during autolysis. The data provide indications of differences in the enzymatic activities confined to different parts of the potato tuber at different physiological stages. Results suggest that native enzymes and substrate proteins of potato tubers can be utilized in search of dietary tools to manage elevated blood pressure.     

Potato extract (Potein) suppresses food intake in rats through inhibition of luminal trypsin activity and direct stimulation of cholecystokinin secretion from enteroendocrine cells

Dietary proteins and trypsin inhibitors are known to stimulate the secretion of the satiety hormone cholecystokinin (CCK). A potato extract (Potein) contains 60% carbohydrate and 20% protein including trypsin inhibitory proteins. In this study, we examined whether Potein suppresses food intake in rats and whether it directly stimulates CCK secretion in enteroendocrine cells. In fasted rats, food consumption was measured up to 6 h after the oral administration of Potein or soybean trypsin inhibitor (SBTI). CCK-releasing activities of Potein and SBTI were examined in the murine CCK-producing cell line STC-1. Potein inhibited the trypsin activity in vitro with a potency 20-fold lower than that of SBTI. Oral administration of Potein dose-dependently suppressed food intake for 1-6 h. Potein, but not the SBTI, dose-dependently induced CCK secretion in STC-1 cells. These results suggest that Potein suppresses food intake through the CCK secretion induced by direct stimulation on enteroendocrine cells and through inhibition of luminal trypsin.     

Angiotensin I-converting enzyme inhibitory peptides in a hydrolyzed chicken breast muscle extract

The blood pressure of spontaneously hypertensive rats (SHRs) decreased after oral administration of an extract prepared from chicken breast muscle, falling maximally to 50 mmHg lower than before. This effect continued for at least 4 h after administration. The peptides possessing hypotensive activity in the chicken extract were examined by measuring the inhibitory activity (IC(50)) against angiotensin I-converting enzyme (ACE). The inhibitory activity of the chicken extract was 1060 mg%, whereas the activity of the extract treated with an Aspergillus protease and gastric proteases (trypsin, chymotrypsin, and intestinal juice) became stronger, reaching 1.1 mg%. Peptides in this hydrolysate of the extract were isolated by HPLC on a reversed-phase column, and their N-terminal sequences were analyzed. Three peptides possessed a common sequence, Gly-X-X-Gly-X-X-Gly-X-X, which was homologous with that of collagen. The peptide Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe showed the strongest inhibitory activity (IC(50) = 42 microM).     

Bowman-Birk and Kunitz Protease Inhibitors among Antinutrients and Bioactives Modified by Germination and Hydrolysis in Brazilian Soybean Cultivar BRS 133

Soybean contains constituents that have antinutritional and bioactive properties. Enzymatic hydrolysis and germination can enhance the biological activity of these compounds in soybean. The objective of this study was to investigate the effect of germination, Alcalase (protease) hydrolysis, and their combination on the concentrations of antinutritional and bioactive compounds in Brazilian soybean cultivar BRS 133. A combination of germination and Alcalase hydrolysis resulted in the degradation of Bowman-Birk inhibitor (BBI), Kunitz trypsin inhibitor (KTI), and lunasin by 96.9, 97.8, and 38.4%. Lectin was not affected by any of the processing treatments when compared to nongerminated and nonhydrolyzed soy protein extract. Total isoflavones (ISF) and total saponins (SAP) increased by 16.2 and 28.7%, respectively, after 18 h of germination, while Alcalase hydrolysis led to the reduction of these compounds. A significant correlation was found between concentrations of BBI and KTI, BBI and lunasin, BBI and ISF, KTI and lunasin, KTI and ISF, KTI and SAP, lunasin and ISF, and ISF and SAP. Germination and Alcalase hydrolysis interacted in reducing BBI, ISF, and SAP. This study presents a process of preparing soy flour ingredients with lower concentrations of antinutritional factors and with biologically active constituents, important for the promotion of health associated with soybean consumption. In conclusion, 18 h of germination and 3 h of Alcalase hydrolysis is recommended for elimination of protease inhibitors, while bioactives are maintained by at least 50% of their original concentrations.     

Purification and characterization of a trypsin inhibitor from Plathymenia foliolosa seeds

A novel trypsin inhibitor (PFTI) was isolated from Plathymenia foliolosa (Benth.) seeds by gel filtration chromatography on a Sephadex G-100, DEAE-Sepharose, and trypsin-Sepharose columns. By SDSPAGE, PFTI yielded a single band with a M(r) of 19 kDa. PFTI inhibited bovine trypsin and bovine chymotrypsin with equilibrium dissociation constants (K(i)) of 4 x 10(-8) and 1.4 x 10(-6) M, respectively. PFTI retained more than 50% of activity at up to 50 degrees C for 30 min, but there were 80 and 100% losses of activity at 60 and 70 degrees C, respectively. DTT affected the activity or stability of PFTI. The N-terminal amino acid sequence of PFTI showed a high degree of homology with various members of the Kunitz family of inhibitors. Anagasta kuehniella is found worldwide; this insect attacks stored grains and products of rice, oat, rye, corn, and wheat. The velvet bean caterpillar (Anticarsia gemmatalis) is considered the main defoliator pest of soybean in Brazil. Diatraea saccharalis, the sugar cane borer, is the major pest of sugar cane crops, and its caterpillar-feeding behavior, inside the stems, hampers control. PFTI showed significant inhibitory activity against trypsin-like proteases present in the larval midguts on A. kuehniella and D. saccharalis and could suppress the growth of larvae.