Study of the Proteolytic Activity of the Tropical Legume 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

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Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Canadian Journal of Microbiology ◽

10.1139/w05-114 ◽

2006 ◽

Vol 52 (1) ◽

pp. 16-23 ◽

Cited By ~ 29

Author(s):

José de Jesús Serrano-Luna ◽

Isaac Cervantes-Sandoval ◽

Jesús Calderón ◽

Fernando Navarro-García ◽

Victor Tsutsumi ◽

...

Keyword(s):

Protease Activity ◽

Serine Proteases ◽

Biochemical Characterization ◽

Acanthamoeba Castellanii ◽

Cysteine Proteases ◽

Skin Lesions ◽

Sds Page ◽

Serine Protease Family ◽

Proteolytic Activities ◽

Amoebic Keratitis

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS–PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to in hibit crude extract protease activity on Madin–Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.Key words: Acanthamoeba spp., amoebic keratitis, serine proteases, cysteine proteases, cytopathic effect.

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

Canadian Journal of Microbiology ◽

10.1139/m94-017 ◽

1994 ◽

Vol 40 (2) ◽

pp. 106-112 ◽

Cited By ~ 9

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.

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

Journal of Insects as Food and Feed ◽

10.3920/jiff2015.0031 ◽

2015 ◽

Vol 1 (4) ◽

pp. 271-280 ◽

Cited By ~ 1

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.

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Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652007000600009 ◽

2007 ◽

Vol 49 (6) ◽

pp. 385-388 ◽

Cited By ~ 4

Author(s):

Erica Boarato David ◽

Semíramis Guimarães ◽

Paulo Eduardo Martins Ribolla ◽

Silvana Torossian Coradi ◽

Diego Peres Alonso

Keyword(s):

Proteolytic Activity ◽

Serine Proteases ◽

High Performance ◽

Giardia Duodenalis ◽

Protein Profiles ◽

Aspartic Proteases ◽

Sds Page ◽

Host Parasite

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

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Serine proteases in the extracellular preparations of Phytophthora infestans: does their presence relate to the aggressiveness of the pathogen?

Plant Protection Science ◽

10.17221/10329-pps ◽

2002 ◽

Vol 38 (SI 1 - 6th Conf EFPP 2002) ◽

pp. S102-S103

Author(s):

J. Hamill ◽

C. Selby ◽

L.R. Cooke

Keyword(s):

Phytophthora Infestans ◽

Serine Protease ◽

Ethyl Ester ◽

Proteolytic Activity ◽

Protease Activity ◽

Serine Proteases ◽

Daily Basis ◽

Serine Protease Activity ◽

Zoospore Suspension

In this study the aggressiveness of nine isolates of P. infestans was determined using detached leaflets from cultivars Bintje and Stirling. The growth of the isolates on the leaflets was recorded on a daily basis, for seven days, and an assessment of their aggressiveness could then be made. Extracellular preparations (ECPs) from the zoospore suspension of each isolate were used as a source of proteolytic activity. The ECPs were found to contain a level of serine protease activity using BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester) as a substrate and recording the absorbance at 256 nm. The possible relationship between the serine protease activity and the aggressiveness of the isolate is discussed.

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Proteolytic compartmentalization and activity in the midgut of Andrallus spinidens Fabricius (Hemiptera: Pentatomidae)

Journal of Entomological and Acarological Research ◽

10.4081/jear.2013.e8 ◽

2013 ◽

Vol 45 (1) ◽

pp. 8 ◽

Cited By ~ 8

Author(s):

S. Sorkhabi-Abdolmaleki ◽

A. Zibaee ◽

H. Hoda ◽

R. Hosseini ◽

M. Fazeli-Dinan

Keyword(s):

Proteolytic Activity ◽

Serine Proteases ◽

Alimentary Canal ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cysteine Proteases ◽

Chloromethyl Ketone ◽

Small Peak ◽

Lepidopteran Larvae ◽

Andrallus Spinidens

Digestive proteolytic activity in the alimentary canal of Andrallus spinidens, a potential biocontrol agent of lepidopteran larvae, was studied by considering enzyme compartmentalization and diversity. The alimentary canal of adults consists of a foregut, a four- sectioned midgut, namely V1 to V4 (ventriculus), and a hindgut. The optimal pH for general proteolytic activity was found to be at pH 8 with a small peak at pH 6. Results revealed that there are several specific proteases in the midgut of A. spinidens, including trypsin-like, chymotrypsin-like, and elastase as serine proteases, and cathepsins B, L and D as cysteine proteases, in addition to two exopeptidases of carboxy- and aminopetidases. Compartmentalization of digestive proteolytic activity showed that V3 is the main area of proteolytic secretion for both general and specific proteases and that V4 has the lowest enzymatic role, so that four out of the eight specific proteases found showed no activity in this section. The lowest and the highest proteolytic activity was found to be in the 1st and 4th nymphal instars, respectively. Using the specific inhibitors phenylmethylsulfonyl fluoride, Na-p-tosyl-L-lysine chloromethyl ketone, Ntosyl- L-phenylalanine chloromethyl ketone, L-trans-epoxysuccinyl-leucylamido-( 4-guanidino)-butane, cystatin, phenanthroline and ethylendiamidetetraacetic acid, we verified the presence of all specific proteases noted using both biochemical assays and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Our findings demonstrated that A. spinidens could utilize several caterpillars because of the presence of various of proteases in its midgut.

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Profile of the body surface proteolytic systém in Apis mellifera quee

Czech Journal of Animal Science ◽

10.17221/150/2009-cjas ◽

2011 ◽

Vol 56 (No. 1) ◽

pp. 15-22 ◽

Cited By ~ 6

Author(s):

A.J. Strachecka ◽

M.M. Gryzińska ◽

M. Krauze ◽

K. Grzywnowicz

Keyword(s):

Protease Inhibitor ◽

Honey Bee ◽

Proteolytic Activity ◽

Body Surface ◽

Protease Activity ◽

Serine Proteases ◽

Developmental Stages ◽

Cysteine Proteases ◽

The Body

The proteolytic system on the body surface of the honey bee has been insufficiently researched. In this study the body surface proteolytic activity was examined in queens at various developmental stages (eggs, larvae, pupae and imagines) in different seasons (spring, summer, autumn, winter). Extracts of the body surface material with water and detergent were used for an in vitro analysis of the proteolytic activity and protease inhibitor level assaying, as well as for an electrophoretic separation of the extracts in polyacrylamide gels. The following methods were used: protein content testing by the Lowry method (modified by Schacterle-Pollack), protease activity testing by the Anson method and protease inhibitor activity testing by the Lee and Lin method. Our studies revealed a high protease activity in an acidic environment (pH = 2.4; the material rinsed with detergent), as well as in neutral (pH = 7) and alkaline (pH = 11.2) environments (the material rinsed with water in both cases). The highest protein concentration values were observed in the imagines from summer. The lowest activities of the proteases and protease inhibitors were determined in the eggs from summer. The highest activities of the acidic, neutral and alkaline proteases were observed in the pupae from spring. The highest number of protease activity bands in PAGE zymography was obtained for the neutral and alkaline activities in the queens for all the seasons. In the queens all the catalytic protease types were present: asparagine and cysteine proteases at pH = 2.4; cysteine proteases and metalloproteases at pH = 7 and serine proteases at pH = 11.2. These results were crucial for the analysis of immunity mechanisms on the body surface of the honey bee.

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Partial protease activity characterization of squid (Todaropsis eblanae) mantle / Caracterización parcial de la actividad proteolítica del manto de pota (Todaropsis eblanae)

Food Science and Technology International ◽

10.1177/108201329900500504 ◽

1999 ◽

Vol 5 (5) ◽

pp. 391-396 ◽

Cited By ~ 16

Author(s):

M.G. Ayensa ◽

H. An ◽

M.C. Gómez-Guillén ◽

P. Montero ◽

A.J. Borderías

Keyword(s):

Cathepsin B ◽

Protease Activity ◽

Cysteine Proteases ◽

Acidic Ph ◽

Alkaline Proteases ◽

Alkaline Ph ◽

Serine Protease Activity ◽

Lysosomal Cysteine Proteases ◽

Ph Range

Proteolytic activity in mantle of Todaropsis eblanae was maximum at 40 and 65 °C. Several peaks of activity were detected over the pH range studied (1.5-9.5), indicating the presence of acidic, neutral and alkaline proteases, depending on the temperature. The substantial enzymic inhibition at acidic pH by the inhibitor trans-epoxysuccinyl-L-leucylamine-4-guanidine butane (E-64) revealed the pre dominance of lysosomal cysteine proteases (cathepsins) which showed higher activity at 65 °C than at 40 °C. At 65 °C and pH 5.5 metallo-proteases were also detected by the inhibition with phenanthroline. Serine protease activity predominated at neutral pH (higher at 40 °C than at 65 °C), and cysteine proteases were detected at alkaline pH. There was evidence of cathepsin B and L activity at 65 °C and to a lesser degree at 40 °C.

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Stem Bromelain Proteolytic Machinery: Study of the Effects of its Components on Fibrin (ogen) and Blood Coagulation

Protein and Peptide Letters ◽

10.2174/0929866527666200525163622 ◽

2020 ◽

Vol 27 (11) ◽

pp. 1159-1170

Author(s):

Mohamed Azarkan ◽

Mariana Marta González ◽

Rafaèle Calvo Esposito ◽

María Eugenia Errasti

Keyword(s):

Blood Coagulation ◽

Proteolytic Activity ◽

Fibrinolytic Activity ◽

Cysteine Proteases ◽

Antithrombotic Agent ◽

Proteolytic System ◽

Total Extract ◽

Low Concentrations ◽

Fibrin Plate ◽

Stem Bromelain

Background: Antiplatelet, anticoagulant and fibrinolytic activities of stem bromelain (EC 3.4.22.4) are well described, but more studies are still required to clearly define its usefulness as an antithrombotic agent. Besides, although some effects of bromelain are linked to its proteolytic activity, few studies were performed taking into account this relationship. Objective: We aimed at comparing the effects of stem bromelain total extract (ET) and of its major proteolytic compounds on fibrinogen, fibrin, and blood coagulation considering the proteolytic activity. Methods: Proteolytic fractions chromatographically separated from ET (acidic bromelains, basic bromelains, and ananains) and their irreversibly inhibited counterparts were assayed. Effects on fibrinogen were electrophoretically and spectrophotometrically evaluated. Fibrinolytic activity was measured by the fibrin plate assay. The effect on blood coagulation was evaluated by the prothrombin time (PT) and activated partial thromboplastin time (APTT) tests. Effects were compared with those of thrombin and plasmin. Results: Acidic bromelains and ananains showed thrombin-type activity and low fibrinolytic activity, with acidic bromelains being the least effective as anticoagulants and fibrinolytics; while basic bromelains, without thrombin-like activity, were the best anticoagulant and fibrinolytic proteases present in ET. Procoagulant action was detected for ET and its proteolytic compounds by the APTT test at low concentrations. The measured effects were dependent on proteolytic activity. Conclusion: Two sub-populations of cysteine proteases exhibiting different effects on fibrin (ogen) and blood coagulation are present in ET. Using well characterized stem bromelain regarding its proteolytic system is a prerequisite for a better understanding of the mechanisms underlying the bromelain action.

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