Studies on Proteolytic Enzyme of the Cricket,Gryllulus taiwanemma Ohmachi

A protease was isolated from the alimentary canal of crickets. This cricket protease was purified by ammonium sulfate, rivanol and acetone fractionations, and DEAE-cellulose and CM-cellulose (Ca-form) column chromatographies. The optimum temperature was 50°C and the optimum pH was 8.0. For preservation, the enzyme was most stable at pH 3.0. Aluminum had the best stabilizing action with no drops of enzyme activity after 24 hours of dialysis. The cricket protease was specific for the synthetic substrate, α-benzoyl-l-arginine amide. Cricket protease had a K_m. 10³ of 2.8 which is similar to that of trypsin, 1.2. The Ea was 3,770 while that of trypsin was 14,960 using α-benzoyl-l-arginine amide as the substrate. Although cricket protease has the same substrate specificity and similar optimum pH and pH-stability as trypsin, it differed in metal requirements to obtain activity. Certain metals are essential for cricket protease activity.     

Behavioural and physiological responses of grass grub larvae (Costelytra zealandica) feeding on protease inhibitors

Abstract

Growth of first instar Costelytra zealandica larvae was significantly reduced after 6 weeks when reared on an artificial diet containing 0.3 and 1% soybean trypsin inhibitor (SBTI), 0.1% and 0.3% potato inhibitor II, and 0.3% potato protease inhibitor I and cowpea trypsin inhibitor. Limabean trypsin inhibitor at 1% significantly stimulated growth compared with that on diet with corresponding levels of added casein. A direct relationship between increased free-trypsin activity and decreased larval growth was observed. Sequential measurement of enzyme activity in third instar larvae feeding on SBTI was compared with that of larvae feeding on casein. The increase in enzyme activity was observed after 14 days in larvae feeding on SBTI. Larvae preferred to feed on SBTI-free diet when given a choice between diet containing this inhibitor at 0.3% and added casein at 0.3%.     

Trypsin Activity Reduced by an Autocatalytically Produced Nonapeptide

Abstract

Trypsin, a serine protease enzyme plays a pivotal role in digestion and is autocatalytic. The crystal structure of a complex formed between porcine trypsin and an auto catalytically produced peptide is reported here. This complex shows a reduction in enzyme activity as compared to native β-trypsin. The nonapeptide has a lysine, which is recognized by Asp 189 at the specificity pocket. The auto catalytically produced native nonapeptide is bound at the active site cleft like other trypsin inhibitors but the important interactions with the oxyanion hole are absent. The peptide covers only a part of the active site cleft and hence the enzyme activity is reduced rather than being inhibited.     

Gold nanomaterials as key suppliers in biological and chemical sensing,catalysis, and medicine

BackgroundGold nanoparticles (AuNPs) with unique physicochemical properties have received a great deal of interest in the field of biological, chemical and biomedical implementations. Despite the widespread use of AuNPs in chemical and biological sensing, catalysis, imaging and diagnosis, and more recently in therapy, no comprehensive summary has been provided to explain how AuNPs could aid in developing improved sensing and catalysts systems as well as medical settings.Scope of reviewThe chemistry of Au-based nanosystems was followed by reviewing different applications of Au nanomaterials in biological and chemical sensing, catalysis, imaging and diagnosis by a number of approaches, and finally synergistic combination therapy of different cancers. Afterwards, the clinical impacts of AuNPs, future application of AuNPs, and opportunities and challenges of AuNPs application were also discussed.Major conclusionsAuNPs show exclusive colloidal stability and are considered as ideal candidates for colorimetric detection, catalysis, imaging, and photothermal transducers, because their physicochemical properties can be tuned by adjusting their structural dimensions achieved by the different manufacturing methods.General significanceThis review provides some details about using AuNPs in sensing and catalysis applications as well as promising theranostic nanoplatforms for cancer imaging and diagnosis, and sensitive, non-invasive, and synergistic methods for cancer treatment in an almost comprehensive manner.     

Purification of Rat Urinary Kallikrein: Comparative Studies with Rat Submandibular Gland Kallikrein-Like Serine Protease

Abstract

A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.     

Isolation and in vitro synthesis of trypsin from the biting fly,Stomoxys calcitrans

The synthesis of [3H]trypsinlike enzyme by the fat body was followed in Stomoxys calcitransin vitro using a radioimmunoassay (RIA) developed against mammalian trypsin. Using high specific activity [3H]valine, trypsinlike activity was followed in midgut epithelial cells, thoracic muscle, and fat body removed from sugar-fed flies. Excreta protease of S. calcitrans was partially purified using charge and hydroxylapatite gel chromatography. Seventy-five percent of the enzyme eluted from these gels was inhibited by tosyl-L-lysine chloromethyl ketone HCI (TLCK) and was classified as trypsinlike. Electrophoresis of the trypsinlike enzyme indicated that it was only 50% pure. Trypsinlike activity from S. calcitrans bound to α₁-globulin IV-I and formed a complex that was dissociated on a P-100 Bio-Gel column. Binding between the protease and the α₁-gobulin IV-I caused a 1.4-fold increase in the apparent molecular weight of the protease on the P-100 Bio-Gel column. Trypsinlike activity was characterized in the midgut and excreta by affinity binding to covalently linked TLCK and tosyl-L-lysine chloromethyl ketone HCI (TAME)Sepharose 4B gels. Between 50% and 55% of the excreta protease and 5669% of the midgut protease bound to the affinity gels and was trypsinlike. Protease activity that did not bind to the gels was not inhibited by TLCK and did not have the esterolytic activity of trypsin.     

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml^–1 of digitonin and 64 µg ml^–1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml^–1 of protease, whereas activity strongly decreases in the presence of 64 µg ml^–1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.     

Induction of motility of apyrene spermatozoa and dissociation of Eupyrene sperm bundles of the silkmoth,Bombyx mori,by initiatorin and trypsin

Summary

The activator, or inducer of motility, of apyrene spermatozoa of the silkmoth, Bombyx mori, was shown to be present in the posterior glandula (g.) prostatica. The activity of this factor in this gland was lost by boiling at 100°C for 10 min. Bundles of eupyrene sperm were dissociated by treatment with either a g. prostatica homogenate or trypsin, and their dissociation was concentration-dependent. On the contrary, for activation of apyrene sperm, the optimal trypsin concentration was 0.45–1.80 μg/ml and activation decreased at higher and lower trypsin concentrations. Microscopic observation showed that the dissociation rate of eupyrene sperm bundles by the g. prostatica homogenate corresponded to that by 0.45–9.0 μg/ml of trypsin. An autolysate of the g. prostatica and digests of seminal fluid with the g. prostatica homogenate or trypsin did not activate apyrene sperm. Of 11 endopeptidase inhibitors tested, antipain, leupeptin, TLCK, TPCK and PMSF strongly inhibited sperm activation by the g. prostatica homogenate, suggesting that the activator is an endopeptidase of the wine protease type. The 6 exopeptidase inhibitors tested did not inhibit activation of apyrene spermatozoa and the dissociation of eupyrene sperm bundles are both caused by the same factor, initiatorin, an endopeptidase of the serine protease type present in the prostatic secretion.     

Acute stress increases colonic paracellular permeability in mice through a mast cell-independent mechanism: Involvement of pancreatic trypsin

AimsIncreased colonic paracellular permeability (CPP) is a key feature of gastro-intestinal disorders as irritable bowel syndrome and inflammatory bowel diseases. Stress stimulates exocrine pancreatic secretion through cholinergic pathways, and trypsin is known to increase CPP. Consequently we have investigated in this work whether trypsin released into the gut lumen following an acute stress may participate to the short-term increase in CPP.Main methodsMice were treated with atropine or a non-selective CRF (corticotropin-releasing factor) receptor antagonist (α-helical CRF (9–41)), before being submitted to a 2-h stress session. Then, CPP and protease activity in colonic contents (total proteolytic, trypsin activity, and mouse mast cell protease (MMCP)-1 levels) were determined. The effects of colonic contents from sham-stressed or stressed animals on CPP were evaluated in mice colonic tissues mounted in Ussing chambers, in presence or not of soybean trypsin inhibitor (SBTI) or FSLLRY, a protease-activated receptor-2 (PAR2) antagonist.Key findingsAcute stress significantly increased CPP, proteolytic and trypsin activities, and MMCP-1 levels. Atropine inhibited stress-induced impairment of CPP and strongly diminished total proteolytic and trypsin activities in stressed animals, but not MMCP-1 levels. Colonic contents from stressed animals increased CPP in mice tissues, this effect being inhibited by SBTI and PAR2 antagonist.SignificanceAcute stress activates cholinergic pathways, to trigger exocrine pancreatic secretion. Trypsin, released in these conditions, may be responsible for colonic barrier alterations through the activation of PAR2.     

Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na⁺-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional protein domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.     

Monte Carlo studies in Gold Nanoparticles enhanced radiotherapy: The impact of modelled parameters in dose enhancement

PurposeOver the last decades, Gold Nanoparticles (AuNPs) have been presented as an innovative approach in radiotherapy (RT) enhancement. Several studies have proven that the irradiation of tumors containing AuNPs could lead to more effective tumor control than irradiation alone. Studies with low kV photons and AuNPs conclude in encouraging results regarding the level of radioenhancement. However, experimental and theoretical studies with MV photons report controversial findings concerning the correlation between dose enhancement effect and tumor cell killing. The great variation in the experimental protocols and simulations complicates the comparison of their outcomes and depicts the need for limiting the variety of investigated parameters. Our purpose is to point out a possible direction for building realistic Monte Carlo (MC) models that could end up with promising results in MV photons RT enhancement.MethodsWe explored published in silico studies concerning AuNPs enhanced RT from 2010 to 2019. In this review, we discuss the different AuNPs and MV photon beams characteristics that have been reported and their effect in dose enhancement.ResultsAuNPs size, concentration, type of distribution along with photon beams energy and the presence of flattening filter in linear accelerators seem to be the major parameters that determine AuNPs radioenhancement in silico.ConclusionsPrior to AuNPs clinical translation in photon radiotherapy, in silico studies should emphasize on nanodosimetry and track structure codes than condensed history ones. Toxicity estimation and biological aspects should be implemented in MC simulations so as to achieve accurate and realistic modelling of AuNPs driven RT.     

Acute inhibition of protease and suppression of growth in zooplankter,Moina macrocopa,by Microcystis blooms collected in Central India

Cyanobacterial blooms consisting of Microcystis spp., collected from 14 water-bodies in Central India, and an adapted culture, were studied for likely impact on zooplankton community. When fed with single cells of Microcystis from several locations, in mixtures with Chlorella, population growth of the cladoceran Moina macrocopa was suppressed. Microcystis alone was unsuitable as food. In three cases, bloom extracts enhanced mortality of starved zooplankton. Extracts from several sources inhibited protease activity when trypsin or a crude extract from zooplankton served as enzyme source. Upon fractionation by solid-phase extraction, the C-18 passed extract contained the anti-protease and toxic substances for zooplankton, whereas a methanol eluted fraction retained the trypsin inhibitory substance. The study suggests that production of protease inhibitors by cyanobacteria is a factor responsible for feeding inhibition and mortality in zooplankton, which in turn could regulate the community structure of grazers.     

Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.

The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of β-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.

The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.

     

Calcium dependent,alkaline detergent-stable trypsin from the viscera of Goby (Zosterisessor ophiocephalus): Purification and characterization

An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was purified to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE. The enzyme had an estimated molecular weight of 23.2 kDa.The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH 7.0 and 11.0. The optimum temperature for enzyme activity was 60 °C, and the activity and stability of trypsin was highly dependent on the presence of calcium ion. At 60 °C, Ca²⁺ (5 mM) stimulated the protease activity by 220%. The trypsin kinetic constants, K_m and k_cat, were 0.312 mM and 2.03 s^?1.The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents.     

In vivo and in vitro effects of wheat germ agglutinin and Bowman-Birk soybean trypsin inhibitor,two potential transgene products,on midgut esterase and protease activities from Apis mellifera

Wheat germ agglutinin (WGA) and Bowman-Birk soybean trypsin inhibitor represent potential transgene products for inducing pest resistance in plants. The effects of these molecules were studied on midgut esterase and protease activities from Apis mellifera L., a major insect pollinator. Trypsin inhibitor and WGA did not exhibit an acute toxicity in A. mellifera. In vivo, trypsin inhibitor caused a decrease in the amount of trypsin activity and did not have a significant effect on esterase activity. In vitro, trypsin inhibitor inhibited about 80% of non-specific protease activity and 100% of trypsin activity. In vivo, WGA at high concentration in food (1 mg/ml) elicited a large decrease in trypsin activity and did not have a significant effect on esterase activity. In vitro, WGA did not have any significant effect on trypsin and non-specific protease activities but slightly activated esterase activity.     

The influence of Li+ ions on pepsin and trypsin activity in vitro

BackgroundThe paper presents a study on the influence of different lithium carbonate and lithium citrate concentration on proteolytic enzymes, namely pepsin and trypsin, in vitro. Lithium can directly affect enzyme activity. Its influence on many bodily functions in both ill and healthy people has been proven.MethodsTo assess the influence of Li⁺ ions concentration and the substrate/enzyme ratio on pepsin and trypsin activity in vitro, 60 factorial experiments were conducted (each repeated 30 times).Main findingsFor both enzymes, statistically significant changes in their activity under the influence of lihium carbonate and lithium citrate were observed. The biggest increase in enzyme activity reached even 198.6 % and the largest decrease in enzyme activity reached about 50 %.ConclusionsThe study shows that both organic and inorganic forms of lithium salts cause changes in the activity of digestive enzymes. Different concentrations of lithium carbonate and lithium citrate stimulate or inhibit the activity of trypsin and pepsin.     

Purification,biochemical, and thermal properties of fibrinolytic enzyme secreted by Bacillus cereus SRM-001

The discovery of microbial fibrinolytic enzymes is essential to treat cardiovascular diseases. This study reports the discovery of a fibrinolytic enzyme secreted by Bacillus cereus SRM-001, a microorganism isolated from the soil of a chicken waste-dump yard. The B. cereus SRM-001 was cultured and the secreted fibrinolytic enzyme purified to show that it is a ～28 kDa protein. The purified enzyme was characterized for its kinetics, biochemical and thermal properties to show that it possesses properties similar to plasmin. A HPLC-MS/MS analysis of trypsin digested protein indicated that the fibrinolytic enzyme shared close sequence homology with serine proteases reported for other Bacillus sp. The results show that the B. cereus SRM-001 secreted enzyme is a ～28 kDa serine protease that possesses fibrinolytic potential.     

Initiation of the degradation of the soybean kunitz and bowman-birk trypsin inhibitors by a cysteine protease

Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti^a, to Ti^a_m, the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the α, α′, and β subunits of soybean β-conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon.     

Estimation of tumor and local tissue dose in gold nanoparticles radiotherapy for prostate cancer

AimThe objective of this research was to estimate the dose distribution delivered by radioactive gold nanoparticles (¹⁹⁸AuNPs or ¹⁹⁹AuNPs) to the tumor inside the human prostate as well as to normal tissues surrounding the tumor using the Monte-Carlo N-Particle code (MCNP-6.1.1 code).BackgroundRadioactive gold nanoparticles are emerging as promising agents for cancer therapy and are being investigated to treat prostate cancer in animals. In order to use them as a new therapeutic modality to treat human prostate cancer, accurate radiation dosimetry simulations are required to estimate the energy deposition in the tumor and surrounding tissue and to establish the course of therapy for the patient.Materials and methodsA simple geometrical model of a human prostate was used, and the dose deposited by ¹⁹⁸AuNPs or ¹⁹⁹AuNPs to the tumor within the prostate as well as to the healthy tissue surrounding the prostate was calculated using the MCNP code. Water and A-150 TEP phantoms were used to simulate the soft and tumor tissues.ResultsThe results showed that the dose due to ¹⁹⁸AuNPs or ¹⁹⁹AuNPs, which are distributed homogenously in the tumor, had a maximal value in the tumor region and then rapidly decreased toward the prostate–tumor interface and surrounding organs. However, the dose deposited by ¹⁹⁸Au is significantly higher than the dose deposited by ¹⁹⁹Au in the tumor region as well as normal tissues.ConclusionsAccording to the MCNP results, ¹⁹⁸AuNPs are a promising modality to treat prostate cancer and other cancers and ¹⁹⁹AuNPs could be used for imaging purposes.     

Phyto-inspired cyclic peptides derived from plant Pin-II type protease inhibitor reactive center loops for crop protection from insect pests

BackgroundNatural defence of plants against insect pests involves protease inhibitors (PIs) that interfere with insect digestive proteases. Pin-II type plant PIs are wound inducible upon insect damage and possess multiple inhibitory repeat domains that can inhibit trypsin and chymotrypsin-like proteases in the insect midgut. Yet, their agricultural ex-vivo application is limited due to large molecular size and environmental instability, which could be overcome by small peptides.MethodsBicyclic peptides were designed by grafting Pin-II PIs derived reactive center loop (RCL) on synthetic tris(bromomethyl)benzene scaffold. In vitro binding with trypsin-like proteases was evaluated by biochemical and biophysical assays, followed by molecular dynamics simulations. In vivo effects on two major lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura were studied upon feeding with peptide treated leaves. Affinity based pull down assays were used to identify target proteins in insect gut.ResultsBicyclic RCLs showed ten-fold enhanced protease inhibition compared to their linear counterparts. They exhibited feeding deterrence and growth reduction of lepidopteran insects. Bicyclic peptides predominantly interact with midgut serine proteases. Possible binding modes involve simultaneous interaction with the active site and specificity-determining residues of insect gut trypsin.ConclusionBicyclic peptides are potent inhibitors of serine proteases in the insect midgut. They cause feeding aversion and larval growth retardation. Bi-domain cyclic peptides interact with two sites on trypsin, leading to enhanced efficacy over linear RCL peptides.General significanceBicyclic peptides mimic natural PIs by inhibiting insect proteases leading to growth reduction, thus, could be used as pest control molecules in agriculture.