Quenched Fluorescent Substrate- Based Peptidase Assays

Lew, Rebecca A.; Tochon-Danguy, Nathalie; Hamilton, Catherine A.; Stewart, Karen M.; Aguilar, Marie‐Isabel; Smith, A. Ian

doi:10.1385/1-59259-877-3:143

Cited by 5 publications

(15 citation statements)

References 3 publications

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“…Conditioned media from HUVECs (60 lL) was added to the specific substrate BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] ; (MW 1907 Da; DIIWVNTPEHVVPYGLG; Auspep, Vic, Australia), or the natural substrate BigET (MW 4283 Da) giving a final concentration of 33 or 66 lg/mL, respectively, in ECE-1 assay buffer. The reaction mixture was incubated at 37°C.…”

Section: Biget and Biget 18-34 Assaymentioning

confidence: 99%

“…All MALDI spectra at time = 0 min confirmed the presence of BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] , but neither the C-terminal cleavage product BigET [22][23][24][25][26][27][28][29][30][31][32][33][34] (MW 1380 Da), nor the N-terminal cleavage product BigET 18-21 (MW 545 Da) were detected (data not shown). BigET [22][23][24][25][26][27][28][29][30][31][32][33][34] was detected in the mixture containing media alone, and in the presence of thiorphan after 2 and 5 h, respectively ( Fig. 3a and b).…”

Section: Biget and Biget 18-34 Assay And Mass Spectrometrymentioning

confidence: 99%

“…The reaction mixture containing BigET and conditioned media alone, as well as in the presence of inhibitors thiorphan/phosphoramidon was also analysed by MALDIToF at time = 0, 1 and 5 h. Spectra at time = 0 confirmed the presence of BigET, while endothelin-1 (ET-1) or the C-terminal cleavage product (BigET [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] ; MW 1808) was not detected (Fig. 4a).…”

Section: Biget and Biget 18-34 Assay And Mass Spectrometrymentioning

confidence: 99%

“…Following reverse phase separation of the reaction mixture consisting of BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] and conditioned media, the fractions containing BigET 18-34 and BigET [22][23][24][25][26][27][28][29][30][31][32][33][34] (m/z 1380) was analysed by tandem mass spectrometry. The amino acid sequence of the resulting peptide fragments and their corresponding molecular masses are listed in Tables 1 and 2.…”

Section: Nano-lc Msms Analysismentioning

confidence: 99%

“…Proteolytic shedding of ECE-1 was measured by monitoring the cleavage of quenched fluorescent substrate (QFS) in conditioned media. A mass spectrometry approach involving BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] , was used to confirm for the first time that ECE-1, endogenously expressed by human umbilical vein endothelial cells undergoes ectodomain shedding. Our data also suggest that ADAM family of metalloproteinases or MMPs are not involved in the ECE-1 ectodomain cleavage.…”

Section: Introductionmentioning

confidence: 99%

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Characterisation of endothelin converting enzyme‐1 shedding from endothelial cells

2007

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The aim of this study was to determine if endothelin converting enzyme-1 (ECE-1) like other members of this metalloprotease family undergoes ectodomain shedding. The release/ shedding of catalytically active ECE-1 was measured by monitoring the fluorescence resulting from the cleavage of a specific quenched fluorescent substrate. Catalytically active ECE-1 was detected in the media of human umbilical vein endothelial cells, and was confirmed by mass spectrometry based assays. Specificity of cleavage was confirmed by using both narrow and broad specificity inhibitors. In conclusion we demonstrate and characterize for the first time, ECE-1 shedding from the surface of endothelial cells.

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Section: Biget and Biget 18-34 Assaymentioning

confidence: 99%

Section: Biget and Biget 18-34 Assay And Mass Spectrometrymentioning

confidence: 99%

Section: Biget and Biget 18-34 Assay And Mass Spectrometrymentioning

confidence: 99%

Section: Nano-lc Msms Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterisation of endothelin converting enzyme‐1 shedding from endothelial cells

2007

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Metallopeptidase inhibition potentiates bradykinin-induced hyperalgesia

Gomez

Por

Berg

et al. 2011

Pain

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The neuropeptide bradykinin (BK) sensitizes nociceptor activation following its release in response to inflammatory injury. Thereafter, the bioactivity of bradykinin is controlled by the enzymatic activities of circulating peptidases. One such enzyme, the metalloendopeptidase EC3.4.24.15 (EP24.15), is co-expressed with bradykinin receptors in primary afferent neurons. In this study, utilizing approaches encompassing pharmacology, biochemistry, cell biology and behavioral animal models, we discover a crucial role for EP24.15 and the closely-related EP24.16 in modulating bradykinin-mediated hyperalgesia. Pharmacological analyses indicate that EP24.15 and EP24.16 inhibition significantly enhances bradykinin type-2 receptor activation by bradykinin in primary trigeminal ganglia cultures. In addition, bradykinin-induced sensitization of TRPV1 activation is increased in the presence of the EP24.15/16 inhibitor JA-2. Furthermore, behavioral analyses illustrate a significant dose-response relationship between JA-2 and bradykinin-mediated thermal hyperalgesia. These results indicate an important physiological role for the metallopeptidases EP24.15 and EP24.16 in regulating bradykinin-mediated sensitization of primary afferent nociceptors.

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N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin

Smith

Rajapakse

Kleifeld

et al. 2016

Sci Rep

Self Cite

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Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer’s disease.

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Quenched Fluorescent Substrate- Based Peptidase Assays

Cited by 5 publications

References 3 publications

Characterisation of endothelin converting enzyme‐1 shedding from endothelial cells

Characterisation of endothelin converting enzyme‐1 shedding from endothelial cells

Metallopeptidase inhibition potentiates bradykinin-induced hyperalgesia

N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin

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