In Vitro Studies on a Proteolytic Enzyme from Aspergillus Oryzae (Protease I)

1968 ◽  
Vol 19 (01/02) ◽  
pp. 136-144 ◽  
Author(s):  
D Ogston ◽  
C. M Ogston
Keyword(s):  
Proteolytic Activity ◽  
Fibrinolytic Activity ◽  
Proteolytic Enzyme ◽  
Thrombolytic Agent ◽  
Inhibitory Effect ◽  
In Vitro Studies ◽  
Clotting Time ◽  
Proteolytic Action ◽  
Fibrinolytic Action

Summary1. Protease I was found to have potent fibrinolytic activity in concentrations which exceeded the blood inhibitory capacity when tested on fibrin plates and artificial thrombi.2. Plasma inhibited the proteolytic activity of protease I to a greater extent than serum; serum had a greater inhibitory effect on protease I than on plasmin. Trasylol did not inhibit the proteolytic action of protease I.3. Protease I caused the slow formation of fibrin in plasma in concentrations which did not produce fibrinogenolysis; this effect was seen in Al(OH)3-adsorbed plasma, and was not inhibited by heparin. Protease I also shortened the recalcified plasma clotting time.4. The fibrinogenolytic action of protease I was more rapid than its fibrinolytic action both in the presence and absence of plasma inhibitors. No concentration of protease I lysed fibrin in plasma without prior destruction or conversion to fibrin of the surrounding plasma fibrinogen.5. It is concluded from these in vitro studies that protease I does not have the properties necessary for a satisfactory thrombolytic agent.

scholarly journals THE ENHANCEMENT OF CHLOROFORM-INDUCED PLASMA PROTEOLYTIC ACTIVITY BY EPSILON AMINOCAPROIC ACID

1962 ◽  
Vol 115 (4) ◽  
pp. 695-706 ◽  
Author(s):  
Virginia H. Donaldson ◽  
Oscar D. Ratnoff
Keyword(s):  
Ionic Strength ◽  
Proteolytic Activity ◽  
Fibrinolytic Activity ◽  
Proteolytic Enzyme ◽  
Aminocaproic Acid ◽  
Heat Stability ◽  
Hydrolytic Activity ◽  
Casein Hydrolysis ◽  
Epsilon Aminocaproic Acid ◽  
Optimal Ph

The proteolytic activity in chloroform-treated plasma euglobulins has been attributed to plasmin. Plasmin can digest both casein and fibrin. Epsilon aminocaproic acid, which inhibits the activation of plasminogen, the precursor of plasmin, by streptokinase, urokinase, and tissue activators enhanced the development of casein hydrolytic activity in a mixture of chloroform and plasma euglobulins. Fibrinolytic activity was also enhanced, but this was evident only if the epsilon aminocaproic acid was removed from the chloroform-treated euglobulins prior to assay. The reasons for the paradoxical enhancement of chloroform-induced casein hydrolysis by euglobulins containing epsilon aminocaproic acid are unclear. However, studies of optimal pH, heat stability, and the effect of ionic strength on the activation of the precursor of this proteolytic enzyme do not differentiate it from plasminogen.

scholarly journals Action of cardosin A from Cynara humilis on ovine and caprine caseinates

2000 ◽  
Vol 67 (3) ◽  
pp. 449-454 ◽  
Author(s):  
SOFIA V. SILVA ◽  
F. XAVIER MALCATA
Keyword(s):  
Proteolytic Activity ◽  
Small Ruminants ◽  
Sodium Caseinate ◽  
Cheese Ripening ◽  
Caprine Milk ◽  
Proteolytic Action ◽  
Milk Coagulation ◽  
Cardosin A ◽  
Intermediate System

In the Iberian Peninsula, the proteinases present in the flowers of members of the Cynara genus, C. cardunculus, C. humilis and C. scolymus, have for many years been successfully used in the manufacture of traditional cheeses from ovine and/or caprine milk on individual farms (Vieira de Sá & Barbosa, 1972; Trujillo et al. 1994). In Portugal, C. cardunculus is the species most frequently employed. Although commercial thistle was tentatively assumed to be pure in taxonomic terms, accurate analyses have shown that the flowers of C. cardunculus are often mixed with flowers of C. humilis (Pires et al. 1994). The clotting activity of C. humilis is due to an aspartic proteinase, currently designated cardosin A and similar to another enzyme obtained from C. cardunculus. This enzyme is similar in specificity and activity to chymosin (Pires et al. 1994).The action of cardosin A from C. cardunculus upon ovine and caprine caseins has been reported recently (Ramalho-Santos et al. 1996; Simo4es, 1998; Sousa & Malcata, 1998), but as yet there is no information on the proteolytic activity of the proteinase from C. humilis upon caseins from milks other than bovine. Caseins from small ruminants' milks are the usual substrates of cardosin during milk coagulation and cheese ripening, and sodium caseinate represents an intermediate system between isolated caseins and the cheese matrix that is free from interference by fat. Thus ovine and caprine caseinates may be useful substrates for investigating the proteolytic activity of cardosin.The aim of the present study was to compare the action of pure cardosin A, obtained from C. humilis, on caprine and ovine caseinates, and to assess the in vitro contribution of this enzyme to the overall proteolytic action of thistle rennet.

scholarly journals Effect of different protein sources on protease activity of northern pike, Esox lucius Linneaus 1758, juvenile

2020 ◽  
Vol 37 (2) ◽  
pp. 135-138
Author(s):  
Cemil Kaya GÖKÇEK ◽  
Tamás SZABÓ ◽  
Cüneyt SUZER
Keyword(s):  
Proteolytic Activity ◽  
Protease Activity ◽  
Fish Meal ◽  
Soybean Protein ◽  
Northern Pike ◽  
Inhibitory Effect ◽  
Live Prey ◽  
Protein Sources ◽  
Corn Gluten

The aim of the study is to determine the inhibitory effect of different protein sources on protease activity of Northern pike, E. Lucius, during larval ontogeny. For this purpose, Northern pike were fed from yolk sac absorption until 21 days after hatching (DAH). At that point, larvae were sampled on 7, 14 and 21 DAH days and the activity of enzyme was analyzed in vitro. In the study, two different fish meal, chicken meal, krill meal, corn gluten, soybean protein concentrate, soybean meal and dried distillers grains with solubles were tested. Fish meal-I showed the lowest effect (7.53 %) on 7 DAH larvae. Moreover, chicken meal has the highest inhibitory effect on the proteases in the first week (68.27%). In the following period (DAH 14), although the inhibition ratio dramatically increased in all ingredients, fish meal-I has still the lowest effect on proteolytic activity (55.66%). In the same period, the highest effect was obtained from krill (82.28 %) and chicken meals (86.73 %), respectively. Then, there was no statistical difference between fish meal-I, fish meal-II and corn gluten in the 21 DAH and relatively lower than the others (p>0.05). Additionally, chicken meal again has the highest effect on juveniles with the ratio 89.27 %. As a result, the increase of proteolytic activity was notably increased in 7-14-21 DAH, however, it is concluded that feeding larvae and juveniles with live prey is still suggested to get better result for such a carnivorous species culture.

scholarly journals Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 3 ◽  
Author(s):  
Chhavi Sharma ◽  
Gad Elsayed Mohamed Salem ◽  
Neha Sharma ◽  
Prerna Gautam ◽  
Rajni Singh
Keyword(s):  
Bacillus Cereus ◽  
Fibrinolytic Activity ◽  
Thrombolytic Agent ◽  
Divalent Cations ◽  
Blood Clot ◽  
Fold Increase ◽  
Protease Production ◽  
Stability Range ◽  
Stain Removal

The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett–Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20–80 °C and pH 5–10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn2+, Zn2+, and Cu2+). The enzyme kinetics revealed Km and Vmax values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI–TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.

scholarly journals Proteolytic Enzyme Production by Strains of the Insect Pathogen Xenorhabdus and Characterization of an Early-Log-Phase-Secreted Protease as a Potential Virulence Factor

2010 ◽  
Vol 76 (20) ◽  
pp. 6901-6909 ◽  
Author(s):  
Mustafa K. Massaoud ◽  
Judit Marokh�zi ◽  
Andr�s Fodor ◽  
Istv�n Venekei
Keyword(s):  
Proteolytic Activity ◽  
Proteolytic Enzyme ◽  
Galleria Mellonella ◽  
Proteolytic Enzymes ◽  
Detection Methods ◽  
Logarithmic Phase ◽  
Bacterial Strains ◽  
Native Page ◽  
Protease B

ABSTRACT As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.

scholarly journals Polyphenols from Chilean Propolis and Pinocembrin Reduce MMP-9 Gene Expression and Activity in Activated Macrophages

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Nicolás Saavedra ◽  
Alejandro Cuevas ◽  
Marcela F. Cavalcante ◽  
Felipe A. Dörr ◽  
Kathleen Saavedra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Proteolytic Activity ◽  
Ethanolic Extract ◽  
Inhibitory Effect ◽  
Ethanol Solution ◽  
Dependent Manner ◽  
Propolis Extract ◽  
Joint Contribution ◽  
Ethanolic Extract Of Propolis

Polyphenols from diverse sources have shown anti-inflammatory activity. In the context of atherosclerosis, macrophages play important roles including matrix metalloproteinases synthesis involved in degradation of matrix extracellular components affecting the atherosclerotic plaque stability. We prepared a propolis extract and pinocembrin in ethanol solution. Propolis extract was chemically characterized using LC-MS. The effect of treatments on gene expression and proteolytic activity was measuredin vitrousing murine macrophages activated with LPS. Cellular toxicity associated with both treatments and the vehicle was determined using MTT and apoptosis/necrosis detection assays. MMP-9 gene expression and proteolytic activity were measured using qPCR and zymography, respectively. Thirty-two compounds were identified in the propolis extract, including pinocembrin among its major components. Treatment with either ethanolic extract of propolis or pinocembrin inhibits MMP-9 gene expression in a dose-dependent manner. Similarly, an inhibitory effect was observed in proteolytic activity. However, the effect showed by ethanolic extract of propolis was higher than the effect of pinocembrin, suggesting that MMP-9 inhibition results from a joint contribution between the components of the extract. These data suggest a potential role of polyphenols from Chilean propolis in the control of extracellular matrix degradation in atherosclerotic plaques.

scholarly journals Antithrombotic potential of Berberis calliobotrys extract

2016 ◽  
Vol 11 (4) ◽  
pp. 776 ◽  
Author(s):  
. Alamgeer ◽  
Hira Asif ◽  
Shahid Rasool
Keyword(s):  
Methanolic Extract ◽  
In Vitro Studies ◽  
Clot Lysis ◽  
Clotting Time ◽  
Blood Samples ◽  
Active Constituents ◽  
Significant Prolongation ◽  
Phytochemical Studies

<p class="Abstract">This study was focused with the aim to investigate the antithrombotic potential of <em>Berberis </em>calliobotrys. Aqueous-methanolic extract and various fractions showed significant (p&lt;0.05-0.001) increase in prothrombin, activated partial thromboplastin and clotting time while only aqueous methanolic extract caused clot lysis when added to the blood samples of rabbit and human. <em>In vivo</em> study in rabbits, butanolic fraction (100 mg/kg) produced more significant prolongation in bleeding, prothrombin, activated partial thromboplastin and clotting time. Interestingly butanolic fraction had shown more pronounced effects among all tested extracts both<em> in vivo</em> and in vitro studies. Hence, it was subjected to antilipid peroxidation and phytochemical studies (total falvonoid contents, HPLC-DAD profile, FTIR). In conclusion, <em>B. </em>calliobotrys induce transient changes in the coagulation parameters, may it possess active constituents responsible for its antithrombotic potential.</p><p> </p>

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