Detecting of fibrino(geno)lytic activity of plant latex proteases by a specific fluorogenic substrate and fibrin(ogen)-zymography

Limulus hemocyte lysate contains a proclotting enzyme, which is transformed to the active clotting enzyme in the presence of gram-negative bacterial endotoxins. The clotting enzyme coagulates a clottable protein, named coagulogen, contained also in the lysate. This gelation reaction of the lysate, named Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. We developed a new fluorogenic substrate, Boc-Leu-Gly-Arg-4-methylcoumarin amide, for Limulus clotting enzyme and established an enzymatic assay method for endotoxins, using the substrate. Because the endotoxin mediates the activation of proclotting enzyme in the lysate, the measurement of amidase activity could be applicable for quantitation of the endotoxins. In fact, the amidase activity determined fluorometrically increased by increasing concentration of E. coli 0111: B4 endotoxin added to the lysate, and a linear relationship between the toxin concentration and the activity was observed in the range of 5X10-6to 5xl0-2 µg endotoxin. The method was a fifty times more sensitive than that of the Limulus test and was very reproducible. However, the method was not directly applicable for the assay of endotoxins in circulating blood, as the amidase activity was strongly inhibited by antithrombin III and α2-plasmin inhibitor. Thus, some pretreatment with heat or chloroform on plasma samples before the assay was required.

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Synthesis of Sulfoquinovose and Sulfoquinovosyl Diacylglycerides, and a Fluorogenic Substrate for Sulfoquinovosidases

10.26434/chemrxiv.10566101.v1 ◽

2019 ◽

Author(s):

Yunyang Zhang ◽

Janice Mui ◽

Thimali Arumaperuma ◽

James P. Lingford ◽

ETHAN GODDARD-BORGER ◽

...

Keyword(s):

Crystal Structures ◽

Fluorogenic Substrate ◽

Potassium Salts ◽

X Ray ◽

Sulfoquinovosyl Diacylglycerol ◽

Sodium And Potassium

The sulfolipid sulfoquinovosyl diacylglycerol (SQDG) and its headgroup, the sulfosugar sulfoquinovose (SQ), are estimated to harbour up to half of all organosulfur in the biosphere. SQ is liberated from SQDG and related glycosides by the action of sulfoquinovosidases (SQases). We report a 10-step synthesis of SQDG that we apply to the preparation of saturated and unsaturated lipoforms. We also report an expeditious synthesis of SQ and (13C6)SQ, and X-ray crystal structures of sodium and potassium salts of SQ. Finally, we report the synthesis of a fluorogenic SQase substrate, methylumbelliferyl a-D-sulfoquinovoside, and examination of its cleavage kinetics by two recombinant SQases.

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Faculty Opinions recommendation of Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717990025.793487077 ◽

2013 ◽

Author(s):

Alain Filloux

Keyword(s):

Vibrio Cholerae ◽

Lytic Activity ◽

Type Vi Secretion

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Antifungal Proteins from Plant Latex

Current Protein and Peptide Science ◽

10.2174/1389203720666191119101756 ◽

2020 ◽

Vol 21 (5) ◽

pp. 497-506

Author(s):

Mayck Silva Barbosa ◽

Bruna da Silva Souza ◽

Ana Clara Silva Sales ◽

Jhoana D’arc Lopes de Sousa ◽

Francisca Dayane Soares da Silva ◽

...

Keyword(s):

Cell Walls ◽

Defense Mechanisms ◽

Hydrolytic Enzymes ◽

Fungal Species ◽

Biologically Active ◽

Protein Classification ◽

Fungal Cell ◽

Antifungal Proteins ◽

Plant Latex

Latex, a milky fluid found in several plants, is widely used for many purposes, and its proteins have been investigated by researchers. Many studies have shown that latex produced by some plant species is a natural source of biologically active compounds, and many of the hydrolytic enzymes are related to health benefits. Research on the characterization and industrial and pharmaceutical utility of latex has progressed in recent years. Latex proteins are associated with plants’ defense mechanisms, against attacks by fungi. In this respect, there are several biotechnological applications of antifungal proteins. Some findings reveal that antifungal proteins inhibit fungi by interrupting the synthesis of fungal cell walls or rupturing the membrane. Moreover, both phytopathogenic and clinical fungal strains are susceptible to latex proteins. The present review describes some important features of proteins isolated from plant latex which presented in vitro antifungal activities: protein classification, function, molecular weight, isoelectric point, as well as the fungal species that are inhibited by them. We also discuss their mechanisms of action.

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Generation Of Factor XII-Dependent Plasminogen Activator Activity In Human Plasma Measured With A Fluorogenic Substrate

10.1055/s-0038-1652135 ◽

1981 ◽

Author(s):

G Dooijewaard ◽

C Kluft

Keyword(s):

Human Plasma ◽

Plasminogen Activator ◽

Minor Role ◽

Factor Xii ◽

Dextran Sulphate ◽

Fluorogenic Substrate ◽

A Value ◽

Substrate Activation ◽

Factor Xiia ◽

A Minor

A rapid fluorometric assay for measurement of amidolytic activity in human plasma was developed, using the plasminogen activator sensitive synthetic substrate t-BOC-L-valyl--glycyl-L-arginine-β-naphthylamide. The plasma is diluted in a reaction cuvet containing 0.050 M Tris HC1 buffer (pH 8.0) and 150 μM substrate. Activation of plasminogen proactivator(s) is initiated at 37°C by the addition of 10 μg dextran sulphate (MW 500,000)/ml. The concentration of β-naphthyl- amide released is recorded fluorometrically as a function of time. The slope of this curve at any time t is proportional to the concentration of activator. Thus, in a single assay, the entire time-dependent profile of activation and subsequent inhibition is monitored; this provides 1. a value for an optimum plasminogen activator content in the plasma, and 2. the time it takes to reach the optimum. The plot of optimum activator content against μl of plasma added is linear for dilutions more than 100-fold, suggesting that under these conditions the optimum content approaches the content of proactivator(s) originally present.The activator content measured predominantly consists of contributions of a factor XII-dependent process since 1. without dextran sulphate or with plasmas deficient in factor XII or prekallikrein no activity could be generated, and 2. plots of optimum activator content against dextran sulphate concentration show sigmoidal-shaped saturation curves as found previously for the kallikrein generation in human plasma. Contributions of factor XIIa and kallikrein only partly account for the content measured and studies with plasmas deficient in factor XI point to a minor role for this factor, if any. Further identification of the activator (s) involved is in progress.

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A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm

Antibiotics ◽

10.3390/antibiotics10030245 ◽

2021 ◽

Vol 10 (3) ◽

pp. 245

Author(s):

Hiroshi Sekiya ◽

Maho Okada ◽

Eiji Tamai ◽

Toshi Shimamoto ◽

Tadashi Shimamoto ◽

...

Keyword(s):

Cell Wall ◽

Clostridium Perfringens ◽

Antimicrobial Agents ◽

Catalytic Domain ◽

Food Poisoning ◽

Binding Activity ◽

Lytic Activity ◽

Intestinal Bacterium ◽

Terminal Domain ◽

Cell Wall Binding

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

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