The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.     

Characterization of digestive proteolytic activity in Lygus hesperus Knight (Hemiptera: Miridae)

The tarnished plant bug, Lygus hesperus Knight, is a pest that causes considerable economic losses to vegetables, cotton, canola, and alfalfa. Detailed knowledge of its digestive physiology will provide new opportunities for a sustainable pest management approach to control this insect. Little is known about the different protease class contributions to the overall digestion of a specific protein. To this end, the proteolytic activities in female adult L. hesperus salivary gland and midgut homogenates were quantified over a range of pH's and time points, and the contribution of different classes of proteases to the degradation of FITC-casein was determined. In the salivary gland, serine proteases were the predominant class responsible for caseinolytic activity, with the rate of activity increasing with increasing pH. In contrast, both aspartic and serine proteases contributed to caseinolytic activity in the midgut. Aspartic protease activity predominated at pH 5.0 and occurred immediately after incubation, whereas serine protease activity predominated at pH 7.5 after a 9h delay and was resistant to aprotinin. The salivary serine proteases were distinctly different from midgut serine proteases, based on the tissue-specific differential susceptibility to aprotinin and differing pH optima. Collectively, the caseinolytic activities complement one another, expanding the location and pH range over which digestion can occur.     

Production of an alkaline protease using Bacillus pumilus D3 without inactivation by SDS,its characterization and purification

Abstract

In this study, protease-producing capacity of Bacillus pumilus D3, isolated from hydrocarbon contaminated soil, was evaluated and optimized. Optimum growing conditions for B. pumilus D3 in terms of protease production were determined as 1% optimum inoculum size, 35?°C temperature, 11 pH and 48?h incubation time, respectively. Stability studies indicated that the mentioned protease was stable within the pH range of 7–10.5 and between 30?°C and 40?°C temperatures. Surprisingly, the activity of the enzyme increased in the presence of SDS with concentration up to 5?mM. The protease was concentrated 1.6-fold with ammonium sulfate precipitation and dialysis. At least six protein bands were obtained from dialysate by electrophoresis. Four clear protein bands with caseinolytic activity were detected by zymography. Dialysate was further purified by anion-exchange chromatography and the caseinolytic active fraction showed a single band between 29 and 36?kDa of reducing conditions.     

Characterization of an alkaline protease from Bacillus sp. no. AH-101

Summary The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0–12.0 respectively. The specific activity toward elastin and keratin was 10 600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes. Offprint requests to: H. Takami     

A survey of proteases in edible mushrooms with synthetic peptides as substrates

The protease activities in six edible mushrooms were surveyed using synthetic fluorogenic substrates that have different specificities for each protease group. The activity was determined by measuring the fluorogenic intensity of the 7-amino-4-methylcoumarin (AMC) liberated by an enzyme. Various types of activities were found in all mushrooms, and their activities depended largely on the mushroom species, but also on the pH and localization. Flammulina velutipes and Pleurotus eryngii had the widest and highest proteolytic activities among the six mushrooms examined. The proteasome-like protease activities were generally much higher than those of other proteases. High caspase activities, which occur during apoptosis in cells, were detected in two mushrooms, F. velutipes and Hypsizigus marmoreus. The pH optima of the proteolytic activities were largely divided into two groups, acidic pH 5–6 for caspases and neutral to alkaline (pH 6.5–11) for the others. In F. velutipes, higher proteolytic activity was observed in the basement of the stem than in the cap and stem. Purification and characterization of protease were also carried out to identify a protease from Grifola frondosa using t-butyloxycarbonyl-Leu-Arg-Arg-4-methylcoumaryl-7-amide (Boc-LRR-MCA) as the substrate.     

Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

THE DETERMINATION OF THE FAR-RED EFFECT IN MARINE PHYTOPLANKTON1,2

A far-red effect exists in 4 marine phytoplankton species: the diatom Ditylum brightwelli, the coccolithophorid Coccolithus huxleyi, the green flagellate Dunaliella tertiolecta, and the dinoflagellate Pyrocystis lunula. The effect is reversible and is manifested through a change in cell division rate. Cultures of algae which received 30-min far-red (FR) light (750 nm) prior to the dark period were compared to controls which received, no FR. Reversal of the FR effect was studied by exposing experimental cultures to 30 min FR followed by 5-min red (R) light (650 nm) prior to the dark period. Controls received only FR. Cultures were exposed to light at 6 different enumerated wavelengths between 460 and 750 nm. A decrease in division rate runs evident only with light at 750 nm. These results give evidence for the presence of the phytochrome system in these phytoplankton species.     

Chymostatin can combine with pepstatin to eliminate extracellular protease activity in cultures of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL-3

Aspergillus strains are being considered as potential hosts for recombinant heterologous protein production because of their excellent extracellular enzyme production characteristics. However, Aspergillus proteases are problematic in that they modify and degrade the heterologous proteins in the extracellular medium. In previous studies we observed that media adjustments and maintenance of a filamentous morphology greatly reduced protease activity and that a low concentration of the aspartic protease inhibitor pepstatin inhibited the latter protease activity to the extent of approximately 90%. In this paper we report that when the serine protease inhibitor chymostatin is used in combination with pepstatin 99–100% of total protease activity in Aspergillus cultures is inhibited. In protease assays a concentration of 30 μM chymostatin combined with 0.075 μM pepstatin was required for maximum inhibition. Inhibitor concentrations of chymostatin and pepstatin of 120 and 0.3 μM, respectively, when added to Aspergillus cultures, has no significant effect on biomass production, glucose utilization or culture pH pattern. The potential of using these protease inhibitors in cultures of recombinant Aspergillus strains producing heterologous proteins will now be investigated to determine if the previously observed recombinant protein denaturing effects of Aspergillus proteases can be negated.     

Differential Growth Responses of Marine Phytoplankton to Herbicide Glyphosate

Glyphosate is a globally popular herbicide to kill weeds and its wide applications may lead to accumulation in coastal oceans as a source of phosphorus (P) nutrient or growth inhibitor of phytoplankton. We studied the physiological effects of glyphosate on fourteen species representing five major coastal phytoplankton phyla (haptophyta, bacillariophyta, dinoflagellata, raphidophyta, and chlorophyta). Based on growth responses to different concentrations of glyphosate under contrasting dissolved inorganic phosphorus (DIP) conditions, we found that phytoplankton species could be classified into five groups. Group I (Emiliania huxleyi, Skeletonema costatum, Phaeodactylum tricornutum) could utilize glyphosate as sole P-source to support growth in axenic culture, but in the presence of DIP, they were inhibited by both 36-μM and 360-μM glyphosate. Group II (Karenia mikimotoi, Prorocentrum minimum, Dunaliella tertiolecta, Symbiodinium sp., Heterosigma akashiwo and Alexandrium catenella) could not utilize glyphosate as sole P-source to support growth, and in the presence of DIP growth was not affected by 36-μM but inhibited by 360-μM glyphosate. Glyphosate consistently enhanced growth of Group III (Isochrysis galbana) and inhibited Group IV (Thalassiosira weissflogii, Thalassiosira pseudonana and Chattonella marina) regardless of DIP condition. Group V (Amphidinium carterae) exhibited no measurable response to glyphosate regardless of DIP condition. This grouping is not congruent with the phylogenetic relationships of the phytoplankton species suggesting functional differentiation driven by environmental pressure. We conclude that glyphosate could be used as P-source by some species while is toxic to some other species and yet has no effects on others. The observed differential effects suggest that the continued use of glyphosate and increasing concentration of this herbicide in the coastal waters will likely exert significant impact on coastal marine phytoplankton community structure.     

Purification and Characterization of Six Kiwifruit Proteases Isolated with Two Ion-exchange Resins,Toyopearl-SuperQ and Bakerbond WP-PEI

Kiwifruit (Actinidia chinensis) contains a cysteine protease, actinidin, and it was suggested to contain two components, A1 and A2. However, the separation of two components was not shown, and the comparison of the two components has not been thoroughly done.

We have now shown that actinidin can be separated into six proteases, named KP1, KP2, KP3, KP4, KP5, and KP6, by improved polyacrylamide gel electrophoresis at pH 4.0. Each kiwifruit protease was purified with two ion-exchange resins, Toyopearl-SuperQ and Bakerbond WP-PEI. Before the purification of kiwifruit proteases, excess p-chloromercuribenzoate was added to crude kiwifruit protease to prevent the autodigestion.

Each kiwifruit protease had a molecular mass of 23,500 and the same amino terminal sequences from the first to the thirteenth. They had different pI’s. These six kiwifruit proteases were divided into two groups by the effects of DTT and Zn²⁺ on the activity.

These results indicated that the six components must be A1, A2, and four previously unknown proteases. Thus we have separated the kiwifruit proteases which were thought to be two, into six components.     

Production of neutral and alkaline extracellular proteases by the thermophilic fungus, Scytalidium thermophilum, grown on microcrystalline cellulose

Extracellular proteases produced by Scytalidium thermophilum, grown on microcrystalline cellulose, were most active at pH 6.5–8 and 37–45 °C when incubated for 60 min. Highest protease activity was at day 3 where endoglucanase activity was low. Protease activity measurements with and without the protease inhibitors, p-chloromercuribenzoate, PMSF, antipain, E-64, EDTA and pepstatin A, suggest production of thiol-containing serine protease and serine proteases. Endoglucanase and Avicel-adsorbable endoglucanase activity in culture medium was not significantly affected by protease inhibitors.     

In-vitro anti-inflammatory activity of serine protease inhibitor from Cassia siamea and Dolichos biflorus: A comparative study

Cassia siamea is a nonedible legume belonging to Fabaceae. The seed of C. siamea contains ~16% of protein. The study reports the biochemical characterization of purified novel serine protease inhibitor from seeds of C. siamea, aimed with assessing the anti-inflammatory activity. The seed extract was subjected to ammonium sulfate precipitation followed by fast protein liquid chromatography (FPLC)-anion exchange chromatography and affinity-chromatography to obtain a relative pure protease inhibitor. Thirty-fivefold purification with the specific activity of 250 U/mg of trypsin inhibitory unit was obtained. The characterization of protease inhibitor for optimum temperature, pH, and metal ions were measured using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) assay and casein zymogram. The C. siamea trypsin inhibitor (CsTI) has a relative molecular mass of 25.540 kDa. Purified CsTI and Dolichos biflorus were tested for anti-inflammatory efficacy against A549 and RAW264.7 cell lines. The inhibitory activity of both purified inhibitors are comparable and are potent toward anti-inflammatory activity. The purified inhibitor shows to be a promising candidate as anti-inflammatory agent by targeting the serine proteases.     

Production of dissolved organic carbon in phyloplankton cultures as measured by high-temperature catalytic oxidation and ultraviolet photo-oxidation methods

The production of dissolved organic carbon (DOC) in culturesof the diatoms Chaetoceros gracilis and Phaeodactylum tricornutum,the flagellate Isochrysis galbana, the dinoflagellate Alexandriumtamarense and a natural algal assemblage from the NorthwestArm, Halifax, Nova Scotia, Canada, was followed using a high-temperaturecatalytic oxidation (HTCO) and a UV photo-oxidation method.Molecular weight fractionation of the DOC was performed fortwo cultures: C.gracilis and I.galbana. While the DOC in theculture medium increased significantly during log-phase growthfor all organisms except the dinoflagellate, this increase wasproportional to the increase in cell numbers; the increase inDOC per cell was either small or zero. In all cultures, maximumrelease took place during stationary and senescent phases, usuallyafter cell numbers had started to decrease. In both C.gracilisand I.galbana, a major portion (>65%) of the organic matterreleased to the medium during log-phase growth had mol. wtsof <10 000 Da. The increase in DOC in the I.galbana culturein stationary and senescent phases was due to the release ofhigh-molecular-weight materials. The differences in extracellularrelease of DOC between species and between different growthstages in the same species suggest that both the species compositionand physiological state of phytoplankton populations must beknown before interpretations and predictions based on fielddata can be made. In order to determine whether the differencesin DOC values found by the HTCO and UV oxidation methods arecaused by the resistance to UV oxidation of some compounds producedby phytoplankton, rather than by less than optimum efficiencyof the UV unit used, standards must be based on naturally occurringcompounds, rather than the pure compounds normally used.     

Purification and characterization of a neutral serine protease with nematicidal activity from <Emphasis Type="Italic">Hirsutella rhossiliensis</Emphasis>

Serine protease plays an important role in fungal infection to invertebrate hosts. An extracellular protease (Hnsp) was detected in liquid culture of Hirsutella rhossiliensis OWVT-1 with nematodes (Panagrellus redivivus) as the unique nitrogen source and purified to homogeneity by ammonium sulphate precipitation, anion exchange chromatography and gel filtration. Its molecular mass was about 32 kDa, and the optimal reaction pH value and temperature were pH 7 and 40°C, respectively. The Hnsp activity was stable at pH 6–8 and decreased radically at 50°C for 10 min. Hnsp was highly sensitive to inhibitor of PMSF and well decomposed the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, suggesting that it belonged to the chymotrypsin/subtilisin of serine proteases. The N-terminal amino acid sequence of Hnsp was SVTDQQGADCGLARISHRE, which showed high homology with other serine proteases from nematophagous fungi. Ability to kill nematode and degrade its cuticle in vitro indicated that Hnsp could be involved in the infection of nematode.     

Protease inhibitors from jackfruit seed (Artocarpus integrifolia)

Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose, xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate oxidation also decreased the inhibitory activity.     

Inhibition of Acid Proteases by a Pepsin Inhibitor (S-PI)

S–PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.

S–PI made an equimolar complex with these acid proteases. A part of the enzyme-S–PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S–PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S–PI free) enzyme, each of the enzyme-S–PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10^?10M, Rh. glutinis acid protease; 1.8×10^?7M, Cladosporium acid protease; 3.0×10^?6M.

These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S–PI complex. S–PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.

Binding between these acid proteases and S–PI dissociated at around neutral pH. S–PI was separated from enzyme-S–PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S–PI complex by DEAE cellulose column chromatography as a native form.     

Prophenoloxidase activation in the hemolymph of Sarcophaga bullata larvae

Phenoloxidase in the hemolymph of Sarcophaga bullata larvae is present as an inactive proenzyme form. Localization studies indicate that the majority of the prophenoloxidase is present in the plasma fraction whereas only a minor fraction (about 4%) is present in the cellular compartments (hemocytes). Inactive prophenoloxidase can be activated by zymosan, not by either endotoxin or laminarin. This activation process is inhibited by the serine protease inhibitors, benzamidine and p-nitrophenyl-p～-guanidobenzoate. Exogenously added proteases, such as chymotrypsin and subtilisin, also activated the prophenoloxidase in the whole hemolymph but failed to activate the partially purified proenzyme. However, an activating enzyme isolated from the larval cuticle, which exhibits trypsinlike specificity, activated the partially purified prophenoloxidase. Inhibition studies and activity measurements also revealed the presence of a similar activating enzyme in the hemolymph. Thus, the phenoloxidase system in Sarcophaga bullata larval hemolymph seems to be comprised of a cascade of reactions. An endogenous protease inhibitor isolated from the larvae inhibited chymotrypsin-mediated prophenoloxidase activation but failed to inhibit the cuticular activating enzyme-catalyzed activation. Based on these studies, the roles of prophenoloxidase, endogenous activating proteases, and protease inhibitor in insect immunity are discussed.     

Immunolocalization and characterization of two novel proteases in Leishmania donovani: Putative roles in host invasion and parasite development

Two novel intracellular proteases having identical molecular mass (58 kDa) were purified from virulent Indian strain of Leishmania donovani by a combination of aprotinin-agarose affinity chromatography, ion exchange chromatography and finally continuous elution electrophoresis. Both of these proteases migrate in SDS-PAGE as a single homogeneous bands suggesting monomeric nature of these proteases. The enzyme activity of one of the proteases was inhibited by serine protease inhibitor aprotinin and another one was inhibited by metalloprotease inhibitor 1, 10 phenanthroline. The purified enzymes were thus of serine protease (SP-Ld) and metalloprotease (MP-Ld) type. The optimal pH for protease activity is 8.0 and 7.5 for SP-Ld and MP-Ld respectively. The temperature optimum for SP-Ld is 28 °C and for MP-Ld is 37 °C showing their thermostability upto 60 °C. Broad substrate (both natural and synthetic) specificity and the effect of Ca²⁺ upon these enzymes suggested novelty of these proteases. Kinetic data indicate that SP-Ld is of trypsin like as BAPNA appears to be the best substrate and MP-Ld seems to be collagenase type as it degrades azocoll with maximum efficiency. Both immunofluorescence and immune-gold electron microscopy studies revealed that the SP-Ld is localized in the flagellar pocket as well as at the surface of the parasite, whereas MP-Ld is located extensively near the flagellar pocket region. This work also suggests that the uses of anti SP-Ld and anti MP-Ld antibodies are quite significant in interfering with the process of parasite invasion and multiplication respectively. Thus the major role of SP-Ld could be predicted in invasion process as it down regulates the phagocytic activity of macrophages, and MP-Ld appears to play important roles in parasitic development.     

The role of viruses in the dynamics of phytoplankton blooms

Abstract

Viruses and virus-like particles (VLP) have been found, in most cases perchance, in about 20 marine phytoplankton species. However only in six of these have there been further investigations on this phenomenon. Different mechanisms of interaction have been hypothesized. These include continuous dynamic viral control on populations (Synechococcus spp.), at times suppressed by external environmental factors (Aureococcus anophagefferens), termination of a bloom caused by viral infection (Emiliania huxleyi and Micromonas pusilla) or by induction of lysogenic cells (Heterosigma akashiwo), and effects on the survivability of specific clones through genetic control (Aureococcus anophagefferens). These examples illustrate the complexity of virus-algae relationships and provide an indication that they may represent a key factor in the dynamics of phytoplankton blooms.