Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

2006 ◽  
Vol 52 (1) ◽  
pp. 16-23 ◽  
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.

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

scholarly journals Profile of the body surface proteolytic systém in Apis mellifera quee

2011 ◽  
Vol 56 (No. 1) ◽  
pp. 15-22 ◽  
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.

ENTOMOPOXVIRUSES ASSOCIATED WITH GRASSHOPPERS AND LOCUSTS: BIOCHEMICAL CHARACTERIZATION

1997 ◽  
Vol 129 (S171) ◽  
pp. 131-146 ◽  
Author(s):  
M.A. Erlandson ◽  
D.A. Streett
Keyword(s):  
Molecular Weight ◽  
Protease Activity ◽  
Biochemical Characterization ◽  
Molecular Techniques ◽  
Enzyme Analysis ◽  
Virus Infections ◽  
Detection And Identification ◽  
Sds Page ◽  
Virus Isolates

AbstractEntomopoxviruses (EPVs) are large DNA viruses with structural similarities to vertebrate poxviruses. EPV virions are occluded in large (3–15 μm in diameter) proteinaceous occlusion bodies (OBs). To date, EPVs are reported from 15 species of grasshoppers and locusts. The current information on the biochemical characterization of these EPVs is summarized in our review. The DNA genomes of grasshopper and locust EPVs analysed to date have a G+C ratio of approximately 18.5% and genome size estimates generated by various methods range from 180 to 194 kilobase pairs (kbp). Restriction endonuclease enzyme analysis of a number of grasshopper and locust EPV DNAs shows the virus isolates to be distinct and the technique will be useful in identifying virus isolates. The structural proteins of certain grasshopper EPVs have also been analysed. Forty to 50 polypeptides ranging in molecular weight from 12 to 200 kilodaltons (kDa) have been detected by SDS-PAGE analysis of virions released from OBs and the polypeptide profiles are distinct for many of the virus isolates. The proteinaceous matrix of the OB of EPVs contains one predominant protein referred to as spheroidin. The spheroidin protein from most grasshopper EPVs is approximately the same molecular weight, 107 kDa, when analysed by SDS-PAGE. As with other groups of occluded insect viruses, grasshopper EPVs have a protease activity associated with OBs derived from infected insects. The possible role of this protease activity in the infection cycle is discussed. Finally, the role of various molecular techniques for the detection and identification of EPV infections in laboratory and field populations of grasshoppers and locusts is discussed. The development of EPV-specific monoclonal antibodies and DNA hybridization probes for the detection of virus infections is reviewed. As well, the possible use of polymerase chain reaction and randomly amplified polymorphic DNA fingerprinting techniques for the detection and identification of EPV infections is discussed.

scholarly journals Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 582
Author(s):  
Cristian Franco-Servín ◽  
Edgar Neri-Castro ◽  
Melisa Bénard-Valle ◽  
Alejandro Alagón ◽  
Ramsés Alejandro Rosales-García ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Biochemical Characterization ◽  
Two Dimensions ◽  
Size Exclusion ◽  
Muscle Twitch ◽  
Amino Terminal ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Main Components

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30–35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve–hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

scholarly journals Reversed Proteolysis—Proteases as Peptide Ligases

Catalysts ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 33
Author(s):  
Peter Goettig
Keyword(s):  
Serine Proteases ◽  
Academic Research ◽  
Building Blocks ◽  
Peptide Bond ◽  
Cysteine Proteases ◽  
Small Peptides ◽  
Natural Peptide ◽  
Peptide Esters ◽  
Natural Amino Acids

Historically, ligase activity by proteases was theoretically derived due to their catalyst nature, and it was experimentally observed as early as around 1900. Initially, the digestive proteases, such as pepsin, chymotrypsin, and trypsin were employed to perform in vitro syntheses of small peptides. Protease-catalyzed ligation is more efficient than peptide bond hydrolysis in organic solvents, representing control of the thermodynamic equilibrium. Peptide esters readily form acyl intermediates with serine and cysteine proteases, followed by peptide bond synthesis at the N-terminus of another residue. This type of reaction is under kinetic control, favoring aminolysis over hydrolysis. Although only a few natural peptide ligases are known, such as ubiquitin ligases, sortases, and legumains, the principle of proteases as general catalysts could be adapted to engineer some proteases accordingly. In particular, the serine proteases subtilisin and trypsin were converted to efficient ligases, which are known as subtiligase and trypsiligase. Together with sortases and legumains, they turned out to be very useful in linking peptides and proteins with a great variety of molecules, including biomarkers, sugars or building blocks with non-natural amino acids. Thus, these engineered enzymes are a promising branch for academic research and for pharmaceutical progress.

N-Terminal amino acid sequence of rat tonin: homology with serine proteases

1978 ◽  
Vol 56 (9) ◽  
pp. 920-925 ◽  
Author(s):  
N. G. Seidah ◽  
R. Routhier ◽  
M. Caron ◽  
M. Chrétien ◽  
S. Demassieux ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Terminal Sequence ◽  
Renin Substrate ◽  
Amino Terminal ◽  
Single Polypeptide Chain ◽  
Serine Protease Family ◽  
Terminal Amino ◽  
Single Polypeptide ◽  
Carboxy Terminal ◽  
Amino Terminal Sequence

In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residue s of the single polypeptide chain composed of 272 amino acids. The se results showed an extensive homology with the sequence of many serine proteases of the trypsin–chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family.

Metalloproteases and egg-hatching in Pediculus humanus, the body (clothes) louse of humans (Phthiraptera: Insecta)

Parasitology ◽  
2007 ◽  
Vol 135 (1) ◽  
pp. 125-130 ◽  
Author(s):  
V. M. BOWLES ◽  
A. R. YOUNG ◽  
S. C. BARKER
Keyword(s):  
Protease Activity ◽  
The Body ◽  
Egg Hatching ◽  
Egg Hatch ◽  
Pediculus Humanus ◽  
Sds Page ◽  
Metalloprotease Inhibitors ◽  
Egg Shell ◽  
Ph Range

SUMMARYTo investigate the biochemical components of egg-hatch in the body louse, Pediculus humanus, egg-shell-washings (ESW) were collected during the first 2 h post-hatching and analysed by gelatin SDS-PAGE. These ESW contained proteases with molecular mass in the range of 25–100 kDa; the most abundant proteases were ~25 kDa. The 3 main regions of protease activity in the one-dimensional gelatin SDS-PAGE gels resolved to at least 23 distinct regions of protease activity when analysed by two-dimensional gelatin SDS-PAGE, with iso-electric points spread over the entire 3 to 10 pH range. Mechanistic characterization indicated that the ESW contained proteases of the metallo-class, inhibited by both 1,10-phenanthroline and EDTA. Several protease inhibitors were tested for their ability to inhibit louse egg-hatch in vitro. The metalloprotease inhibitor 1,10-phenanthroline and the aminopeptidase inhibitor bestatin significantly inhibited (P<0·05) louse egg-hatch (100% and 58%, respectively). The presence of metalloproteases at the time of egg-hatch and the inhibition of egg-hatch in P. humanus by metalloprotease inhibitors suggests a crucial role for these proteases in the hatching of this medically important parasite.

scholarly journals Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

2011 ◽  
Vol 435 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Pitter F. Huesgen ◽  
Helder Miranda ◽  
XuanTam Lam ◽  
Manuela Perthold ◽  
Holger Schuhmann ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Protein Quality ◽  
Biochemical Characterization ◽  
Pdz Domain ◽  
Control Mechanisms ◽  
Ph Optimum ◽  
Periplasmic Proteins ◽  
Synechocystis Sp ◽  
Pcc 6803

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.

scholarly journals Inhibition of Staphylococcus aureus cysteine proteases by human serpin potentially limits staphylococcal virulence

2011 ◽  
Vol 392 (5) ◽  
Author(s):  
Tomasz Kantyka ◽  
Karolina Plaza ◽  
Joanna Koziel ◽  
Danuta Florczyk ◽  
Hennig R. Stennicke ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Serine Proteases ◽  
Peptide Bond ◽  
Cysteine Proteases ◽  
Reactive Site ◽  
Association Rate ◽  
High Association ◽  
Endogenous Protease ◽  
Covalent Complex

AbstractBacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing thatStaphylococcus aureuscysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (kass) for inhibitory complex formation (1.9×104m/s and 5.8×104 m/s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activityin vivoat epithelial surfaces infected/colonized byS. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of nativeS. aureus-derived inhibitors of the staphopains (staphostatins). Given thatS. aureusis a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.

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