Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 x 10(-9) M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

A Kunitz proteinase inhibitor from Archidendron ellipticum seeds: purification, characterization, and kinetic properties

Leguminous plants in the tropical rainforests are a rich source of proteinase inhibitors and this work illustrates isolation of a serine proteinase inhibitor from the seeds of Archidendron ellipticum (AeTI), inhabiting Great Nicobar Island, India. AeTI was purified to homogeneity by acetone and ammonium sulfate fractionation, and ion exchange, size exclusion and reverse phase chromatography (HPLC). SDS-PAGE of AeTI revealed that it is constituted by two polypeptide chains (alpha-chain, M(r) 15,000 and beta-chain, M(r) 5000), the molecular weight being approximately 20 kDa. N-terminal sequence showed high homology with other serine proteinase inhibitors belonging to the Mimosoideae subfamily. Both Native-PAGE as well as isoelectric focussing showed four isoinhibitors (pI values of 4.1, 4.55, 5.27 and 5.65). Inhibitory activity of AeTI remained unchanged over a wide range of temperatures (0-60 degrees C) and pH (1-10). The protein inhibited trypsin in the stoichiometric ratio of 1:1, but lacked similar stoichiometry against chymotrypsin. Also, AeTI-trypsin complex was stable to SDS unlike the SDS unstable AeTI-chymotrypsin complex. AeTI, which possessed inhibition constants (K(i)) of 2.46 x 10(-10) and 0.5 x 10(-10)M against trypsin and chymotrypsin activity, respectively, retained over 70% of inhibitory activity after being stored at -20 degrees C for more than a year. Initial studies on the insecticidal properties of AeTI indicate it to be a very potent insect anti-feedant.     

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.     

A serine proteinase inhibitor from frog eggs with bacteriostatic activity

By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.     

Trypsin inhibitors of buffalo seminal plasma.

Two trypsin inhibitors from acid-treated buffalo seminal plasma were purified by gel filtration and affinity chromatography. These acid-stable trypsin inhibitors having charge heterogeneity were homogeneous with respect to size as revealed by gel filtration and SDS-PAGE. Gel filtration data suggest molecular weight value of 9,900 Da for inhibitor I and 10,900 Da for inhibitor II. Molecular weight estimated by SDS-PAGE was found to be 10,600 Da and 11,200 Da for inhibitors I and II, respectively. The hydrodynamic properties such as Stokes radii (1.58 nm and 1.62 nm); intrinsic viscosity (2.5725 ml/g and 2.5025 ml/g) and diffusion coefficient (13.499 x 10(-11) m2/sec. and 13.166X10(-11) m2/sec) respectively for inhibitor I and II were determined by analytical gel filtration. These inhibitors were fairly thermostable and could not be stained by PAS reagent. Both the inhibitors were found to inhibit buffalo acrosin but not bovine chymotrypsin.     

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.     

A novel trypsin inhibitor from Peltophorum dubium seeds,with lectin-like properties,triggers rat lymphoma cell apoptosis

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.     

Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.     

Trypsin Inhibitor from Poecilanthe parviflora Seeds: Purification, Characterization, and Activity Against Pest Proteases

Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves of being attacked by insects. In this work, a novel trypsin inhibitor (PPTI) was purified from the seeds of the native Brazilian tree Poecilanthe parviflora (Benth) (Papilioinodeae, Leguminosae) by gel filtration chromatography on a Sephadex G-100 followed by Superdex G75 chromatography (FPLC), Sepharose 4B-Trypsin column, and fractionated by reversed-phase HPLC on a C-18 column. SDS-PAGE showed that PPTI consisted of a single polypeptide chain with molecular mass of about 16 kDa. The dissociation constant of 1.0 x 10(-7) M was obtained with bovine trypsin. PPTI was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the PPTI showed a high degree of homology with other Kunitz-type inhibitors. Trypsin-like activity in midguts of larval Diatraea saccharalis, Anagasta kuehniella, Spodoptera frugiperda, and Corcyra cephalonica were substantially inhibited by PPTI.     

Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 × 10^{? 9} M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

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.     

A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae)

A novel trypsin inhibitor was purified from the seeds of Peltophorum dubium (Spreng.). SDS-PAGE under reducing conditions showed that the inhibitor consisted of a single polypeptide chain (ca. 20 kDa). The dissociation constants of 4 x 10(-10) and 1.6 x 10(-10) M were obtained with bovine and porcine trypsin, respectively. This constant was lower (2.6 x 10(-7) M) for chymotrypsin. The inhibitory activity was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the P. dubium inhibitor showed a high degree of homology with other Kunitz-type inhibitors. When fed to the insect Anagasta kuehniella, in an artificial diet (inhibitor concentration 1.6%), the inhibitor produced approximately 56% and delayed the development of this lepidopteran. The concentration of inhibitor in the diet necessary to cause a 50% reduction in the weight (ED50) of fourth instar larvae was approximately 1%. The action of the P. dubium trypsin inhibitor (PDTI) on A. kuehniella may involve inhibition of the trypsin-like activity present in the larval midgut, resistance of the inhibitor to digestion by midgut enzymes and bovine trypsin, and association of the inhibitor with a chitin column and chitinous structures in the peritrophic membrane and/or midgut of the insect.     

Characterization of urinary proteinase inhibitors with segments of amino acids sequences identical to sequences of pancreatic secretory trypsin inhibitor

1. Slow migrating proteinase inhibitors were isolated from pathological human urine. 2. The N-terminal amino acid sequence including 23 amino acids was identical to the one in pancreatic secretory trypsin inhibitor. 3. The slow migrating proteinase inhibitors occurred in 3 forms with different electrophoretic mobility. 4. Time of flight mass spectrometry showed that the Mw of one of the forms was 6241 while the Mw of another form was 5923. 5. The Ki of complexes with trypsin was determined to be 1 x 10(-10) M, with chymotrypsin and plasmin Ki was 1 x 10(-7) M. Elastase, kallikrein and thrombin were not inhibited.     

Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties

A trypsin inhibitor from Dimorphandra mollis seeds was isolated to apparent homogeneity by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange and affinity chromatographic techniques. SDS-PAGE analysis gave an apparent molecular weight of 20 kDa, and isoelectric focusing analysis demonstrated the presence of three isoforms. The partial N-terminal amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz family of inhibitors. This inhibitor, which inhibited trypsin activity with a Ki of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site.     

Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10^–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.     

Proteinase inhibitors from the excretory gland cells of Stephanurus dentatus. Purification and properties of three secretory proteinase inhibitors

Three proteinase inhibitors designated as I, II, and III were isolated from the excretory gland cells of the swine kidney worm, Stephanurus dentatus. The inhibitors, which were trichloroacetic acid-soluble, were purified by affinity chromatography and ion exchange chromatography. The homogeneity of each inhibitor was shown by polyacrylamide gel electrophoresis and electrofocusing. The molecular weights of the inhibitors estimated by sodium dodecyl sulfate gel electrophoresis fell within a limited range of 9300 to 9700, and the isoelectric points were 6.45, 6.20, and 5.34 for Inhibitors I, II, and III, respectively. The inhibitors formed complexes with trypsin having apparent dissociation constants (Ki) of 2.9 X 10(-11), 7.6 X 10(-11), and 6.4 X 10(-11) M, respectively. Each inhibitor inhibits the esterolytic and proteolytic activities of both trypsin and chymotrypsin. A proteinase inhibitor present in the reproductive organs, intestines, body walls, and esophagi was identical with Inhibitor II found in the excretory gland cells. Culture medium collected after 24-h incubation with adult worms contained the same three inhibitors as the excretory gland cells. These data suggest that the gland cells may secrete the inhibitors internally and externally.     

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.     

Induction of a proteinase K inhibitor in a potato cultivar with a high degree of field resistance against Phytophthora infestans

A proteinase K inhibitor (PLPKI) was isolated from a potato cultivar with a high level of field resistance ( Solanum tuberosum L. cv. Pampeana INTA), after 24 h of infection with Phytophthora infestans , when inhibitory activity was markedly increased. Purification was performed by heat treatment, gel filtration chromatography and affinity chromatography. A size of 60 kDa was estimated by SDS-PAGE in partially denaturing conditions and by gel filtration. It is multimeric and the monomer has a molecular mass of 8.5–9.0 kDa. PLPKI is highly active against proteinase K (EC 3.4.21.14) but poorly inhibits two serine proteinases of animal origin, trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). A differential expression (determined by activity and immunoblotting assays) of PLPKI was observed between two potato cultivars with different degrees of field resistance to P. infestans . In the resistant cultivar (cv. Pampeana INTA) PLPKI induction (19-fold with respect to healthy leaves) occurred 24 h after infection and remained over basal levels after 48 h infection. By contrast, in the susceptible cultivar (cv. Bintje), no induction was observed.     

Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis

A screening assay for inhibitory activity against trypsin in skin mucus from 29 species of fishes reveals a wide distribution of trypsin inhibitors in skin mucus and relatively high antitryptic activity in pufferfish of the family Tetraodontidae. Two trypsin inhibitors termed TPTI 1 and 2 were purified to homogeneity from the skin mucus of Takifugu pardalis by salting out, lectin affinity, anion exchange FPLC and gel filtration HPLC. Both inhibitors are acidic glycoproteins, with an apparent molecular mass of 57 kDa in SDS-PAGE, pI below 4 and 1.9% reducing sugar for TPTI 1 and with an apparent molecular mass of 47 kDa in SDS-PAGE, pI 5.2 and 0.8% reducing sugar for TPTI 2. The inhibitors effectively repress the catalytic activity of trypsin and alpha-chymotrypsin, and therefore can be classified as serine protease inhibitors. The inhibitory constants against trypsin were 4.9x10(-8) M for TPTI 1 and 3.9x10(-8) M for TPTI 2. Both inhibitors react with trypsin at a molar ratio of 1:1, although TPTI 1 reversibly inactivates the proteolytic activity of trypsin non-competitively and TPTI 2, competitively. The trypsin inhibitors in the skin mucus of T. pardalis may function as defense substances to neutralize serine proteases released by invasive pathogens.