Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

Study of the interaction of trypsin inhibitor from the sea anemone Radianthus macrodactylus with proteases

The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: K_D = 7.38 × 10^?8 M and 9.93 × 10^?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively.     

Trypsin inhibition by Ligupurpuroside B as studied using spectroscopic,CD, and molecular docking techniques

It is well known that Ligupurpuroside B is a water-soluble polyphenolic compound and used to brew bitter tea with antioxidant activities. It acted as a stimulant to the central nervous system and a diuretic (increase the excretion of urine), was used to treat painful throat and high blood pressure, and also exerted weight-loss function. In this regard, a detailed investigation on the mechanism of interaction between Ligupurpuroside B and trypsin could be of great interest to know the pharmacokinetic behavior of Ligupurpuroside B and for the design of new analogues with effective pharmacological properties. Ligupurpuroside B successfully quenched the intrinsic fluorescence of trypsin via static quenching mechanism. The binding constants (K_a) at three temperatures (288, 298, and 308 K) were 1.7841?×?10⁴, 1.6251?×?10⁴ and 1.5483?×?10⁴ L mol^?1, respectively. Binding constants revealed the stronger binding interaction between Ligupurpuroside B and trypsin. The number of binding sites approximated to one, indicating a single class of binding for Ligupurpuroside B in trypsin. The enzyme activity result suggested that Ligupurpuroside B can inhibit trypsin activity. Thermodynamic results revealed that both hydrogen bonds and hydrophobic interactions play main roles in stabilization of Ligupurpuroside B-trypsin complex. Circular dichroism (CD) results showed that the conformation of trypsin changed after bound to ligupurpuroside B. Molecular docking indicated that Ligupurpuroside B can enter the hydrophobic cavity of trypsin and was located near Trp215 and Tyr228 of trypsin.

Communicated by Ramaswamy H. Sarma     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

Biochemical characterisation of a Kunitz-type inhibitor from Tamarindus indica L. seeds and its efficacy in reducing plasma leptin in an experimental model of obesity

A trypsin inhibitor isolated from tamarind seed (TTI) has satietogenic effects in animals, increasing the cholecystokinin (CCK) in eutrophy and reducing leptin in obesity. We purified TTI (pTTI), characterised, and observed its effect upon CCK and leptin in obese Wistar rats. By HPLC, and after amplification of resolution, two protein fractions were observed: Fr1 and Fr2, with average mass of [M?+?14H]⁺?=?19,594,690?Da and [M?+?13H]⁺?=?19,578,266?Da, respectively. The protein fractions showed 54 and 53 amino acid residues with the same sequence. pTTI presented resistance to temperature and pH variations; IC₅₀ was 2.7?×?10^?10?mol.L^?1 and Ki was 2.9?×?10^?11?mol.L^?1. The 2-DE revealed spots with isoelectric points between pH 5 and 6, and one near pH 8. pTTI action on leptin decrease was confirmed. We conclude that pTTI is a Kunitz trypsin inhibitor with possible biotechnological health-related application.     

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.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Characterisation of the endospermal trypsin inhibitor of barley

A trypsin inhibitor was isolated from grains of two row barley (cv. Proctor). The purified protein was identical with the corresponding inhibitor of a six row barley (cv. Pirkka); both proteins showed, a Pi of 7.4. The N-terminal amino acid was phenylalanine and an arginine residue was involved in the active site. Effects of substrate concentration showed that the inhibition was noncompetitive with a K_i of about 0.9 × 10^?7M. An enzyme-inhibitor complex was demonstrated by disc electrophoresis.     

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

Abstract

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10?kDa, respectively, and under non-reducing conditions, 26?kDa was observed. The inhibitor was shown to inhibit bovine trypsin (K_i of 3.45?nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.     

Isolation and characterization of a protease inhibitor from spinach leaves

An acid-stable and heat-labile proteinous protease inhibitor which was found in spinach leaves but not in seeds was isolated by sequential chromatography and preparative isoelectric focusing. The isoelectric point of this inhibitor was 4.5. The inhibitor had a M_r of ca 18 000 and was rich in aspartic acid and glycine; it had 4 half-cystine, 2 tryptophan and no methionine residues. Its extinction coefficient (E|_cm^%) was 13.7 at 280 nm. The inhibition was competitive and the dissociation constant was 3.32 × 10^?13 M. The inhibitor was specific to serine proteases and strongly inhibited trypsin and weakly inhibited α-chymotrypsin and kallikrein.     

N-monoarylacetothioureas as potent urease inhibitors: synthesis,SAR, and biological evaluation

Abstract

A urease inhibitor with good in vivo profile is considered as an alternative agent for treating infections caused by urease-producing bacteria such as Helicobacter pylori. Here, we report a series of N-monosubstituted thioureas, which act as effective urease inhibitors with very low cytotoxicity. One compound (b19) was evaluated in detail and shows promising features for further development as an agent to treat H. pylori caused diseases. Excellent values for the inhibition of b19 against both extracted urease and urease in intact cell were observed, which shows IC₅₀ values of 0.16?±?0.05 and 3.86?±?0.10?µM, being 170- and 44-fold more potent than the clinically used drug AHA, respectively. Docking simulations suggested that the monosubstituted thiourea moiety penetrates urea binding site. In addition, b19 is a rapid and reversible urease inhibitor, and displays nM affinity to urease with very slow dissociation (k _off=1.60?×?10^?3 s^?1) from the catalytic domain.     

Structural insights into the unique inhibitory mechanism of Kunitz type trypsin inhibitor from Cicer arietinum L.

Kunitz-type trypsin inhibitors bind to the active pocket of trypsin causing its inhibition. Plant Kunitz-type inhibitors are thought to be important in defense, especially against insect pests. From sequence analysis of various Kunitz-type inhibitors from plants, we identified CaTI2 from chickpea as a unique variant lacking the functionally important arginine residue corresponding to the soybean trypsin inhibitor (STI) and having a distinct and unique inhibitory loop organization. To further explore the implications of these sequence variations, we obtained the crystal structure of recombinant CaTI2 at 2.8Å resolution. It is evident from the structure that the variations in the inhibitory loop facilitates non-substrate like binding of CaTI2 to trypsin, while the canonical inhibitor STI binds to trypsin in substrate like manner. Our results establish the unique mechanism of trypsin inhibition by CaTI2, which warrant further research into its substrate spectrum. Abbreviations BApNA Nα-Benzoyl-L-arginine 4-nitroanilide

BPT bovine pancreatic trypsin

CaTI2 Cicer arietinum L trypsin inhibitor 2

DrTI Delonix regia Trypsin inhibitor

EcTI Enterolobium contortisiliquum trypsin inhibitor

ETI Erythrina caffra trypsin inhibitor

KTI Kunitz type inhibitor

STI soybean trypsin inhibitor

TKI Tamarindus indica Kunitz inhibitor

Communicated By Ramaswamy H. Sarma     

Temporary site in the anti-tryptic fragment from adzuki-bean proteinase inhibitor II

The anti-tryptic fragment, derived from adzuki-bean proteinase inhibitor II, was subjected to limited proteolysis by trypsin at pH 2.9 for 48 h. Three peptide bonds, Lys-Ser, Arg-Cys and Arg-Asp, were split, inactivating the fragment. The temporary site, the point of inactivation against trypsin, was concluded to be Arg-Cys, since the Lys-Ser bond is the reactive site and the tripeptide (Asp)_3′ released by the cleavage of the Arg-Asp bond, should not affect the inhibitory activity. This effective bond, corresponding to Arg³²-Cys³³ of inhibitor II, was possibly more exposed to the enviromental solvent by cuting down the anti-chymotryptic domain from the parent inhibitor.     

Potential Anticoagulant Activity of Trypsin Inhibitor Purified from an Isolated Marine Bacterium <Emphasis Type="Italic">Oceanimonas</Emphasis> Sp. BPMS22 and its Kinetics

Protease inhibitors control major biological protease activities to maintain physiological homeostasis. Marine bacteria isolated from oligotrophic conditions could be taxonomically distinct, metabolically unique, and offers a wide variety of biochemicals. In the present investigation, marine sediments were screened for the potential bacteria that can produce trypsin inhibitors. A moderate halotolerant novel marine bacterial strain of Oceanimonas sp. BPMS22 was isolated, identified, and characterized. The effect of various process parameters like salt concentration, temperature, and pH was studied on the growth of the bacteria and production of trypsin inhibitor. Further, the trypsin inhibitor was purified to near homogeneity using anion exchange, size exclusion, and affinity chromatography. The purified trypsin inhibitor was found to competitively inhibit trypsin activity with an inhibition coefficient, K_i, of 3.44?±?0.13 μM and second-order association rate constant, k_ass, of 1.08?×?10³ M^?1 S^?1. The proteinaceous trypsin inhibitor had a molecular weight of approximately 30 kDa. The purified trypsin inhibitor showed anticoagulant activity on the human blood samples.     

The inhibitory kinetics and mechanism of glycolic acid on lipase

Abstract

Obesity is prone to cause a variety of chronic metabolic diseases, and it has aroused people’s attention that the rapid increase in the global population of obese people in the past years. As a kind of weight-loss drug acting in the intestine, lipase inhibitor does not enter the bloodstream without producing central nervous side effects. Because they do not affect the metabolism system, lipase inhibitors and obesity have become one of the hot spots in recent years. Glycolic acid is a new substrate analog inhibitor with the value of the semi-inhibitory concentration of lipase is estimated to be 17.29?±?0.14?mM. Using the plots of Lineweaver-Burk, the inhibition mechanism of lipase by glycolic acid was reversible and the inhibition type belongs to competitive inhibition with a K_I value of 19.61?±?0.26?mM. The inhibitory kinetics assay showed that the microscopic velocity constant k₊₀ of inhibition kinetics is 1.79?×?10^?3?mM^?1s^?1, and k_?0 is 0.73?×?10^?3 s^?1. The results of UV full-wavelength scanning on product cumulative, fluorescence quenching and molecular simulation also indicated that glycolic acid and substrate competitive with lipase by binding to Lys137. Thereby glycolic acid inhibiting the oxidation-catalyzed reaction and reducing the product of the enzyme and substrate. This adds a new direction for the search for lipase inhibitors and provides new ideas about the development of anti-obesity drugs.

Communicated by Ramaswamy H. Sarma     

Biochemical characterization and structural analysis of trypsin from Plodia interpunctella midgut: implication of determinants in extremely alkaline pH activity profile

In the present study, trypsin from Plodia interpunctella (Hübner) is characterized to discover sequence, biochemical and structural features. This enzyme is purified by ion exchange chromatography using fast protein liquid chromatography on proteins from fifth‐instar larvae. The enzyme is optimally active at 50 °C and pH 11.0. The kinetic parameters (K_m and V_max) of the enzyme are 5.3 ± 0.6 µm and 31 ± 1.3 nmol min^?1 mg^?1, respectively (using Nα‐benzoyl‐l ‐arginine ρ‐nitroanilide hydrochloride as substrate). The enzyme is inhibited by the addition of Cu²⁺ and Mn²⁺, whereas it is activated by Li⁺ at high concentrations. Moreover, the enzyme is almost completely inhibited in the presence of Nα‐tosyl‐l ‐lysine chloromethyl ketone hydrochloride and phenylmethanesulphonyl fluoride. To understand some characteristics of P. interpunctella trypsin, including active site structure and alkaline pH profile, a reliable structural model of P. interpunctella trypsin is built based on the Fusarium oxisporum (Schlecht) trypsin cystal structure (Protein Data Bank code: 1GDU). The secondary structure content of the purified trypsin from near‐ultraviolet circular dichroism data shows considerable similarities with that of P. interpunctella trypsin predicted structure. Analysis of pK_a values of active site residues, a type of amino acid residue in the active site cleft and the surface charges of the model and Tribolium castaneum (Herbst) trypsin structure as an insect species from different orders reveals some differences between them. These differences might effect on the microenvironment of the active site cleft and consequently shift its pH profile. The application of multiple theoretical and experimental techniques is well adapted to predict the enzyme structure with high accuracy and this could help in the design of a powerful inhibitor for trypsin with ideal properties.     

Inhibition studies of soybean (Glycine max) urease with heavy metals,sodium salts of mineral acids,boric acid,and boronic acids

Various inhibitors were tested for their inhibitory effects on soybean urease. The K_i values for boric acid, 4-bromophenylboronic acid, butylboronic acid, and phenylboronic acid were 0.20?±?0.05?mM, 0.22?±?0.04?mM, 1.50?±?0.10?mM, and 2.00?±?0.11?mM, respectively. The inhibition was competitive type with boric acid and boronic acids. Heavy metal ions including Ag⁺, Hg²⁺, and Cu²⁺ showed strong inhibition on soybean urease, with the silver ion being a potent inhibitor (IC₅₀ = 2.3?×?10^?8 mM). Time-dependent inhibition studies exhibited biphasic kinetics with all heavy metal ions. Furthermore, inhibition studies with sodium salts of mineral acids (NaF, NaCl, NaNO₃, and Na₂SO₄) showed that only F^? inhibited soybean urease significantly (IC₅₀ = 2.9?mM). Competitive type of inhibition was observed for this anion with a K_i value of 1.30?mM.     

Production of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae

The spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media increased the spore yield in solid-state from three to sevenfold: A. clavatus produced 48.4?±?5.2 and 15.7?±?1.6?×?10⁸ spores/g, A. flavus produced 22.3?±?4.1 and 3.1?±?2.5?×?10⁸ spores/g, and M. anisopliae produced 39.6?±?6.5 and 13.1?±?2.6?×?10⁸ spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC₅₀) of 1.1, 1.8, and 1.3?×?10⁸ spores/ml against Culex quinquefasciatus Say larvae in 72?h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognised entomopathogenic fungus.     

Comprehensive optimization of composite cryoprotectant for Saccharomyces boulardii during freeze-drying and evaluation of its storage stability

Abstract

Saccharomyces boulardii (S. boulardii) is widely adopted in the diarrhea treatment for humans or livestock, so guaranteeing the survival rate of S. boulardii is the critical issue during freeze-drying process. In this study, the survival rate of S. boulardii with composite cryoprotectants during freeze-drying procedure and the subsequent storage were investigated. With the aid of response surface method, the composite cryoprotectants were comprehensively optimized to be lactose of 21.24%, trehalose of 22.00%, and sodium glutamate of 4.00%, contributing to the supreme survival rate of S. boulardii of 64.22?±?1.35% with the viable cell number of 9.5?±?0.07?×?10⁹ CFU/g, which was very close to the expected rate of 65.55% with a number of 9.6?×?10⁹ CFU/g. The accelerated storage test demonstrated that the inactivation rate constant of the freeze-dried S. boulardii powder was k_?18?=?8.04?×?10^?6. In addition, the freeze-dried goat milk powder results exhibited that the inactivation rate constants were k₄?=?4.48?×?10^?4 and k₂₅?=?9.72?×?10^?3 under 4 and 25?°C, respectively. This work provides a composite cryoprotectant formulation that has a good protective effect for the probiotic S. boulardii during freeze-drying process, possessing the potential application prospect in food, medicine, and even feed industry.