Some characteristics of extracellular proteases produced by members of the Chytridiales and the Spizellomycetales (Chytridiomycetes)

1994 ◽  
Vol 40 (2) ◽  
pp. 106-112 ◽  
Author(s):  
Thomas Krarup ◽  
Lauritz W. Olson ◽  
Hans Peter Heldt-Hansen
Keyword(s):  
Proteolytic Activity ◽  
Isoelectric Focusing ◽  
Serine Proteases ◽  
Proteolytic Enzymes ◽  
Complex Compounds ◽  
Extracellular Proteases ◽  
Peptide Substrates ◽  
Proteolytic Activities ◽  
Selection Of

The extracellular proteolytic enzymes of eight saprophytic, eucarpic, and monocentric isolates from two genera of the order Spizellomycetales and from one genus of the order Chytridiales (Chytridiomycetes) have been partially characterized. The isolectric points of the proteases were estimated from zymograms and demonstrate the existence of three types of proteolytic activity in most isolates. The proteases were tested against synthetic chromogenic peptide substrates and a selection of cations and more complex compounds, and the results suggest that parts of the extracellular proteolytic activities are due to proteases from two groups: the Ca2+ stabilized proteases and the alkaline serine proteases.Key words: serine proteases, metalloproteases, Chytridiomycetes, isoelectric focusing, chromogenic peptide substrates.

Study of the Proteolytic Activity of the Tropical Legume Crotalaria spectabilis

2012 ◽  
Vol 67 (9-10) ◽  
pp. 495-509 ◽  
Author(s):  
Juliana da Silva Pacheco ◽  
Raquel Elisa da Silva-Lopez
Keyword(s):  
Proteolytic Activity ◽  
Serine Proteases ◽  
Ph Value ◽  
Cysteine Proteases ◽  
Leaf Extracts ◽  
Serine Protease Activity ◽  
Proteolytic Activities ◽  
Low Levels ◽  
Crotalaria Spectabilis

The characterization of legume proteases contributes to the understanding of the physiology of plants and their interaction with the environment. Thirteen extracts from various parts of Crotalaria spectabilis were made using different extraction systems. The highest protein content was found in seeds, and the most pronounced proteolytic activity was observed in leaf extracts, with an optimal pH value in the alkaline range. Proteases in extracts from roots, stems, and fl owers were active in various pH ranges. Proteases in all extracts were maximally active between 30 °C and 60 °C and were thermostable (24 h, 60 °C). Hemoglobin, bovine serum albumin, casein, and gelatin were hydrolyzed by C. spectabilis extracts in different ways. The highest serine protease activity was found in leaves. Seeds contained high levels of serine proteases and low levels of cysteine proteases. Flowers, roots, and stems contained different levels of serine, aspartic, and metalloproteases, respectively. The proteolytic activities in extracts were modulated by cations and oxidants to various degrees. C. spectabilis proteases are differentially expressed in distinctive organs, and their stability against heat and oxidants makes this plant an important source of stable proteases

An initial characterization of the proteolytic enzymes secreted by the adult stage of the human hookworm Necator americanus

Parasitology ◽  
1995 ◽  
Vol 110 (5) ◽  
pp. 555-563 ◽  
Author(s):  
A. Brown ◽  
J. M. Burleigh ◽  
E. E. Billett ◽  
D. I. Pritchard
Keyword(s):  
Serine Proteases ◽  
Metal Ion ◽  
Proteolytic Enzymes ◽  
Maximum Activity ◽  
Biologically Relevant ◽  
Necator Americanus ◽  
Recent Developments ◽  
Proteolytic Activities ◽  
Secretory Products ◽  
Ph Range

SUMMARYThe proteolytic activities present in adult Necator americanus excretory–secretory products have been assessed using biologically relevant, naturally occurring substrates (haemoglobin and fibrinogen) and a number of synthetic fluorogenic and chromogenic substrates. One broad peak of activity was observed against haemoglobin in the pH range 5 to 7, with maximum activity at pH 6·6, while fibrinogenolytic activity was shown to be greater at pH 3·5. Inhibition studies against haemoglobin, fibrinogen and synthetic substrates using a battery of appropriate protease inhibitors indicated the presence of a mixture of aspartyl, cysteinyl and serine proteases. Metal ion (Ca2+, Zn2+ and Fe2+) stimulation was demonstrated, with stimulation by Zn2+ being the most marked. These results are discussed in the context of recent developments in the field of parasite proteolytic enzymes, where they have been suggested as targets for immuno- and chemotherapy.

Synthetic Peptide Assays For Monitoring The Defects Of Extrinsic And Intrinsic Pathways Of Coagulation: Selection Of A Suitable Substrate

1981 ◽  
Author(s):  
Jeanine Walenga ◽  
Jawed Fareed ◽  
Harry L Messmore ◽  
Judith Kniffin
Keyword(s):  
Partial Thromboplastin Time ◽  
Synthetic Peptide ◽  
Serine Proteases ◽  
Developmental Stages ◽  
Peptide Substrate ◽  
Amidolytic Activity ◽  
Proper Selection ◽  
Peptide Substrates ◽  
Free Peptide ◽  
Selection Of

Several assays based on the use of thrombin specific synthetic peptide substrates have been proposed for the amidolytic equivalents of prothrombin time (PT) and partial thromboplastin time (PTT). All of these methods employ the same principle as the clotting assays; the test plasma is activated with either thromboplastin or activator-cephalo-plastin mixture and the generated thrombin activity is measured employing thrombinr specific synthetic substrates such as Bz-Phe-Val-Arg-pNA (S-2160), H-D-Phe-Pip-Arg-pNA (S-2238), Tos-Gly-Pro-Arg-pNA (Chromozym TH), and CH3-Gly- Pro-Arg-pNA. These substrates and their free peptide forms inhibit the amidolytic action of bovine Xa activated human and Xa Russell’s viper venom in the following order: S-2160 > S-2238 > Sarc-Pro-Arg-pNA ≥ Chromozym TH. A new substrate for thrombin Pyro-Glu-Pro-Arg-pNA (S-2366) and a plasminogen activator (tissue) substrate, H-D-Ile-Pro-Arg-pNA (S-2288) have been tested and were found to produce weaker inhibition of bovine and human Xa. Tos-Gly-Pro-Arg-pNA, Sarc-Pro-Arg-pNA and H-D-Ile-Pro-Arg-pNA were found to produce a ≤ 10% inhibition of the activator generated Xa’s amidolytic activity at concentrations which are commonly employed in the PT and PTT assays. Although in the developmental stages the synthetic substrate equivalent assays are more sensitive than the existing PT and PTT assays and provide useful information on the total amount of thrombin generated in each assay, our results suggest that amidolytic equivalent assays for PT and PTT are feasable. However proper selection of a peptide substrate is important as some of the thrombin substrates and their free peptide forms may inhibit Xa and other serine proteases which are generated during the activation step thereby seriously influencing the results.

Structural and physicochemical changes due to proteolytic deterioration of escamoles (Liometopum apiculatum) a traditional Mexican food

2015 ◽  
Vol 1 (4) ◽  
pp. 271-280 ◽  
Author(s):  
A. Castillo-Andrade ◽  
R. García-Barrientos ◽  
M.A. Ruiz-Cabrera ◽  
C. Rivera-Bautista ◽  
J.D. Pérez-Martínez ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Serine Proteases ◽  
Structural Changes ◽  
Iodoacetic Acid ◽  
Initial Structure ◽  
Physicochemical Changes ◽  
Proteolytic Activities ◽  
Food Needs ◽  
Endogenous Proteases

Entomophagy or consumption of insects has significantly increased worldwide, either for pleasure or to satisfy the food needs in developing countries. There are approximately 2,000 species of edible insects distributed in 120 countries. From these 2,000 species, about 540 are located in Mexico; one of the most consumed are the escamoles. Escamoles are larvae and pupae of the ant Liometopum apiculatum. Escamoles are nutritious because of their high content of protein, fat, carbohydrates and vitamin. However, during storage the quality of escamoles changes rapidly which affects the acceptability by the consumer. This loss of quality is probably a result of proteolytic activity of endogenous proteases. Therefore, the objectives of this study were to identify the classes of proteases in escamoles as well as to evaluate the effect of proteolytic activity on physicochemical and structural changes during storage. Proteases identification was conducted using specific inhibitors; structural changes, texture, and proteolytic activity were monitored at different days of storage. The highest proteolytic activities (P<0.05) were observed at pH 8, 9 and 10 and at 37 and 50 °C. Proteases were mainly inhibited by iodoacetic acid and soybean trypsin inhibitor showing that cysteine and serine proteases were dominant. High proteolytic activity, significant (P<0.05) reduction in texture and weight loss was observed during storage. The deterioration of escamoles was evident in analyses of images, where initial structure was lost during storage. These results indicate that different groups of proteases are associated with rapid deterioration of escamoles.

scholarly journals LPS Counter Regulates RNA Expression of Extracellular Proteases and Their Inhibitors in Murine Macrophages

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Andreas Hald ◽  
Birgitte Rønø ◽  
Leif R. Lund ◽  
Kristoffer L. Egerod
Keyword(s):  
Extracellular Matrix ◽  
Matrix Metalloproteinase ◽  
Serine Proteases ◽  
Gene Expression Regulation ◽  
Proteolytic Enzymes ◽  
Matrix Degradation ◽  
Extracellular Proteases ◽  
The Matrix ◽  
Matrix Metabolism ◽  
Temporal Expression Pattern

Besides their evident importance in host defense, macrophages have been shown to play a detrimental role in different pathological conditions, including chronic inflammation, atherosclerosis, and cancer. Regardless of the exact situation, macrophage activation and migration are intimately connected to extracellular matrix degradation. This process is accomplished by multiple proteolytic enzymes, including serine proteases and members of the matrix metalloproteinase family. In this study, we have utilized qPCR arrays to simultaneously analyze the temporal expression pattern of a range of genes involved in extracellular matrix metabolism in the mouse derived-macrophage cell line RAW 264.7 following stimulation with LPS. Our results revealed that LPS induces the expression of matrix metalloproteinases while at the same time decreased the expression of matrix metalloproteinase inhibitors. The opposite scenario was found for the genes encoding serine proteases, which were downregulated while their inhibitors were upregulated. In addition, intergenic comparison of the expression levels of related proteases revealed large differences in their basal expression level. These data highlight the complexity of the gene expression regulation implicated in macrophage-dependent matrix degradation and furthermore emphasize the value of qPCR array techniques for the investigation of the complex regulation of the matrix degradome.

Purification of Oidiodendron kalrai proteases

1976 ◽  
Vol 22 (3) ◽  
pp. 327-333 ◽  
Author(s):  
P. M. Cino ◽  
R. P. Tewari
Keyword(s):  
Ammonium Sulfate ◽  
Proteolytic Activity ◽  
Column Chromatography ◽  
Crude Extract ◽  
Proteolytic Enzymes ◽  
Deae Cellulose ◽  
Ammonium Sulfate Precipitation ◽  
Proteolytic Activities ◽  
Yeast Phase ◽  
Cellulose Column

Oidiodendron kalrai yeast-phase cells demonstrate proteolytic activity. Some of the proteolytic enzymes of the crude extract were purified by a combination of ammonium sulfate precipitation, Sephadex G-200, and diethylaminoethyl (DEAE) cellulose column chromatography. At least six proteins exhibiting a range of proteolytic activities could be identified by these procedures. Purity of the enzyme fractions obtained from the DEAE-cellulose columns was tested by running polyacrylamide gels.

Proteolytic system of the sperm of Apis mellifera drones

Biologia ◽  
2013 ◽  
Vol 68 (3) ◽  
Author(s):  
Grzegorz Borsuk ◽  
Aneta Strachecka ◽  
Krzysztof Olszewski ◽  
Jerzy Paleolog ◽  
Jacek Chobotow ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Proteolytic Activity ◽  
Serine Proteases ◽  
Protein Concentration ◽  
Proteolytic Enzymes ◽  
Electrophoretic Analysis ◽  
Polyacrylamide Gel ◽  
Inhibitor Activity ◽  
Lowry Method ◽  
Triton X

AbstractDuring many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.

An assessment of the contribution of plant proteinases to proteolytic digestion in the rumen of sheep

2000 ◽  
Vol 2000 ◽  
pp. 5-5
Author(s):  
R. J. Wallace ◽  
S. J. A. Wallace ◽  
N. McKain
Keyword(s):  
Proteolytic Activity ◽  
Broad Spectrum ◽  
Dietary Protein ◽  
Proteolytic Enzymes ◽  
Ammonia Production ◽  
Proteolytic Digestion ◽  
Protein Breakdown ◽  
Ruminal Microorganisms ◽  
Rapid Release ◽  
Proteolytic Activities

Protein breakdown in the rumen often leads to excessive ammonia production and inefficient use of dietary protein by ruminants (Wallace et al., 1997). Attention has for many years focussed on the proteolytic activity of ruminal microorganisms (Wallace et al., 1997). The wide variety of proteolytic species and proteolytic enzymes and their between-animal variability has made the task of decreasing microbial proteolytic activity difficult (Falconer & Wallace 1999). Much less attention has been paid to the contribution of proteinases originating from the feed. In particular, grass cells contain vacuoles harbouring broad spectrum proteinases which are known to be responsible for protein breakdown in the silo (Wetherall et al., 1995). Theodorou et al. (1996) proposed that much of the rapid release of ammonia in grazing animals might be initiated by the action of plant, rather than microbial, proteinases. The present study was undertaken to compare the proteolytic activities of fresh grass and ruminal microorganisms and to evaluate their likely contributions to ammonia production in the rumen.

Proteolysis on the body surface of pyrethroid-sensitive and resistant Varroa destructor

2013 ◽  
Vol 58 (1) ◽  
Author(s):  
Aneta Strachecka ◽  
Grzegorz Borsuk ◽  
Krzysztof Olszewski ◽  
Jerzy Paleolog ◽  
Zbigniew Lipiński
Keyword(s):  
Protease Inhibitor ◽  
Proteolytic Activity ◽  
Body Surface ◽  
Serine Proteases ◽  
Protein Concentration ◽  
Proteolytic Enzymes ◽  
Electrophoretic Analysis ◽  
The Body ◽  
Activity Levels

AbstractThe aim of this work was to determine the activity of proteases and protease inhibitors sampled from the body surface of tau-fluvalinate-sensitive and resistant V. destructor. Proteins were isolated from the tau-fluvalinate-sensitive and resistant mites, while mites untreated with tau-fluvalinate constituted the control. Subsequently, the following methodology was applied: protein concentration assay by the Lowry method — as modified by Schacterle and Pollack; assay of proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; identification of proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee and Lin method; identification of acidic, neutral and basic protease activities by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method and for protease inhibitor detection with the Felicioli method. The highest value of protein concentration was found in the tau-fluvalinate-sensitive V. destructor, while the highest activity levels of acidic, neutral and alkaline proteases were observed in the tau-fluvalinate-resistant mites. Aspartic, serine, thiolic and metallic proteases were found in the drug-resistant and drug-sensitive Varroa mites. The control samples were found to contain aspartic and serine proteases. In an acidic and alkaline environment, the results revealed a complete loss of inhibitor activities in the in vitro analyses and electrophoresis. Serine protease inhibitor activities (at pH 7.0) were high, especially in the group of tau-fluvalinate-resistant mites.

Purification and properties of extracellular proteases produced by the nematophagous fungus Verticillium suchlasporium

1990 ◽  
Vol 36 (8) ◽  
pp. 530-537 ◽  
Author(s):  
L. V. Lopez-Llorca
Keyword(s):  
Proteolytic Activity ◽  
Serine Proteases ◽  
Extracellular Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fluorescein Isothiocyanate ◽  
Exchange Chromatography ◽  
Extracellular Proteases ◽  
Cyst Nematodes

The fungal parasite of eggs of cyst nematodes, Verticillium suchlasporium, produced extracellular proteases when grown in semiliquid culture with gelatin as the only source of nitrogen and carbon. The proteolytic activity of culture filtrates was maximum 12–14 days after inoculation. Gel filtration chromatography in Sephadex G-100 resolved two peaks of proteolytic activity. The peak accounting for most of the activity was further purified by ion-exchange chromatography in SP-Sephadex C-25 as a single peak. This protease had a molecular mass of 32 kDa calculated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The enzyme was an endopeptidase that degraded fibrinogen in zymograms and had an optimum pH of 8.5 using fluorescein isothiocyanate – casein as the substrate. It was inhibited by phenylmethylsulfonyl fluoride, indicating that it was a serine protease. The purified protease was able to degrade certain cyst nematode proteins suggesting the involvement and specificity of the 32-kDa protease during nematode egg penetration by Verticillium suchlasporium. Key words: nematophagous fungi, Verticillium suchlasporium, extracellular enzymes, serine proteases.

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