A new fluorogenic substrate for chymotrypsin

Zimmerman, M.D.; Yurewicz, Edward C.; Patel, Gool F.

doi:10.1016/s0003-2697(76)80066-8

Cited by 183 publications

(68 citation statements)

References 5 publications

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“…Because bacteria are known to contain serine proteinases (24,25), we tested various preparations of C. rodentium for the release of trypsin-like activity using Boc-Gln-Ala-Arg-AMC as a substrate (26,27). Incubation of this substrate in the presence of either intact or outer membrane preparations of C. rodentium did not demonstrate trypsin-like activity.…”

Section: Resultsmentioning

confidence: 99%

A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis

Hansen

Sherman²,

Cellars³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

165

156

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Citrobacter rodentium is a bacterial pathogen that causes a murine infectious colitis equivalent to enterohemorrhagic Escherichia coli infection in humans. Colonic luminal fluid from C. rodentiuminfected mice, but not from sham-infected mice, contains active serine proteinases that can activate proteinase-activated receptor-2 (PAR2). We have identified granzyme A and murine trypsins to be present in C. rodentium-infected luminal fluid, as determined by mass spectrometry and Western blot analysis. Inflammatory indices (colonic mucosa macroscopic damage score, increased intestinal wall thickness, granulocyte infiltration, and bacterial translocation from the colonic lumen to peritoneal organs) were all increased in C. rodentium-infected mice, compared with shaminfected mice. Soybean trypsin inhibitor-treated wild-type mice and untreated PAR2-deficient (PAR2 ؊/؊ ) mice (compared with their wild-type littermates) both had substantially reduced levels of C. rodentium-induced inflammation. These data point to an important role for both pathogen-induced host serine proteinases and PAR2 in the setting of infectious colitis.colitis ͉ inflammation ͉ trypsin ͉ granzyme

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Section: Resultsmentioning

confidence: 99%

A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis

Hansen

Sherman²,

Cellars³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

165

156

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“…and mixed anhydride method. (Nedev et al 1993;Pozdnev 1994;Zimmerman et al 1976;Fujiwara and Tsuru 1978;Sharma and Castellino 1990;Okada et al 1982;Rijkers et al 1995;Kato et al 1978;Monatalbetti and Flaque 2005). Due to weak nucleophilicity of arylamines, highly activated carboxylic acid is required for complete conversion into arylamides.…”

Section: Introductionmentioning

confidence: 99%

An Efficient and Epimerization Free Synthesis of C-Terminal Arylamides Derived from α-Amino Acids and Peptide Acids via T3P Activation

Madhu

NageswaraRao

Narendra

et al. 2014

Int J Pept Res Ther

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“…A series of synthetic fluorogenic substrates, specific for a number of serine proteases, utilizing the leaving group 7-amino-4-methylcoumarin (AMC) has been described (20,21). In a continuation of this approach, we have now prepared a synthetic peptide specific for the cleavage site of PA, incorporating the same leaving group.…”

Section: Introductionmentioning

confidence: 99%

“…There is a need, however, for a simple, sensitive, direct assay that allows both rapid measurement and kinetic analysis of PA, independent of plasmin generation. In addition, the presence of two proteases of similar specificities in the two-step assay precludes the screening of potential PA inhibitors.A series of synthetic fluorogenic substrates, specific for a number of serine proteases, utilizing the leaving group 7-amino-4-methylcoumarin (AMC) has been described (20,21). In a continuation of this approach, we have now prepared a synthetic peptide specific for the cleavage site of PA, incorporating the same leaving group.…”

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confidence: 99%

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. (20,21). Activation and emission wavelengths (383 and 455 nm, respectively) were chosen such that AMC retained 20% of its maximal fluorescence but possessed a relative fluorescence 500-fold greater than that of an equimolar amount of Cbz-Gly-Gly-Arg-AMC (Table 1).…”

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confidence: 99%

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Direct fluorescent assay of urokinase and plasminogen activators of normal and malignant cells: kinetics and inhibitor profiles.

Zimmerman¹,

Quigley²,

Ashe³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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A direct rate assay for plasminogen activator has been developed using a synthetic fluorogenic peptide substrate, 7(N-Cbz-glycylglycylargininamido)4-methylcoumarin trifluoroacetate. The assay correlates well with the standard 12SI-labeled fibrin plate assay using highly purified urokinase, culture fluids from WI-38, Chinese The enhanced production of plasminogen activator (PA) activity has been shown to be a characteristic of many different cell types. The intracellular and extracellular levels of PA have been demonstrated to be substantially elevated in malignant cells in culture (1-6), cells treated with a tumor promoter (7), activated macrophages (8, 9), established cell lines (10, 11), granulosa cells during ovulation (12), embryonic cells during differentiation (13,14), and hormone-treated uteri (15). The standard system used for measuring PA in these cells is an indirect, two-step assay in which plasminogen is incubated with a source of PA and the plasmin activity generated is quantitated by using fibrin, casein, or protamine as substrates (16)(17)(18)(19). There is a need, however, for a simple, sensitive, direct assay that allows both rapid measurement and kinetic analysis of PA, independent of plasmin generation. In addition, the presence of two proteases of similar specificities in the two-step assay precludes the screening of potential PA inhibitors.A series of synthetic fluorogenic substrates, specific for a number of serine proteases, utilizing the leaving group 7-amino-4-methylcoumarin (AMC) has been described (20,21). In a continuation of this approach, we have now prepared a synthetic peptide specific for the cleavage site of PA, incorporating the same leaving group. This compound is Cbz-GlyGly-Arg-AMC. We report here the use of this substrate in the direct fluorescent assay of PA from normal and malignant cells, some kinetic parameters of these enzymes, and the effect of various low and high molecular weight protease inhibitors on the various enzymes. The various PAs were analyzed by the direct fluorescent technique in parallel with the indirect standard 25I-labeled fibrin plate assay using purified plasminogens from both canine and bovine sources. (24). When the cultures had attained high cell density (1 X 107 cells per 100-mm culture dish), the plates were washed three times with minimal medium and further incubated in serum-free minimal medium. The medium was removed from the cultures every 12 hr and was a source of extracellular PA; it is referred to as harvest fluid (HF). After it was harvested, the HF was immediately centrifuged to remove cells and cellular debris and acidified to pH 3.5 by the addition of 1 M HCl. Ammonium sulfate was added to 70% saturation, and the resulting precipitate was recovered by centrifugation and resuspended in 1/100 the volume of the original HF in 0.05 M glycine.HCI buffer, pH 3.0. MATERIALS AND METHODSWI-38, HeLa, and Chinese hamster ovary cells were grown in serum-containing medium, washed, and incubated in serum-free Higuchi medium (25). This me...

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A new fluorogenic substrate for chymotrypsin

Cited by 183 publications

References 5 publications

A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis

A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis

An Efficient and Epimerization Free Synthesis of C-Terminal Arylamides Derived from α-Amino Acids and Peptide Acids via T3P Activation

Direct fluorescent assay of urokinase and plasminogen activators of normal and malignant cells: kinetics and inhibitor profiles.

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