Direct photometric or fluorometric assay of proteinases using substrates containing 7-amino-4-trifluoromethylcoumarin

Smith, Robert E.; Bissell, Eugene R.; Mitchell, Ann; Pearson, Keenan

doi:10.1016/0049-3848(80)90074-2

Cited by 94 publications

(30 citation statements)

References 17 publications

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“…The abundance of different C-, N-, and P-degrading enzymes in soils is controlled by numerous factors including microbial biomass, community composition, substrate availability, microclimate, and stoichiometric demands 5,6 . However, in situ EEAs within the soil environment are also affected by temperature 7,8 , the binding of enzymes to soil clays and humic properties 18,19 . MUC-linked substrates are commonly associated with N-rich synthetic substrates such as proteins and/or amino acids.…”

Section: Introductionmentioning

confidence: 99%

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Bell

Fricks

Rocca

et al. 2013

JoVE

278

224

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Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.

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Section: Introductionmentioning

confidence: 99%

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Bell

Fricks

Rocca

et al. 2013

JoVE

278

224

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“…Thus, AFC has been utilized as the fluorophore for protease activity detection. [12] Furthermore, the 19 F NMR spectrum of AFC shows only a singlet peak without any coupling to intramolecular protons. AFC is thus appropriate for 19 F MRI.…”

Section: Methodsmentioning

confidence: 96%

Dual‐Function Probe to Detect Protease Activity for Fluorescence Measurement and ¹⁹F MRI

et al. 2009

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“…Finally, for determination of plasmin activity, t-butoxycarbonyl-Val-Leu-Lys-AMC . AcetylOH (Bachem) was employed as a substrate of the reaction lasting 30 min at 37°C, in 50 mM TES buffer, pH 8.0 [28].…”

Section: Activity Of Glomerular Proteinasesmentioning

confidence: 99%

Mechanism of antinephritic effect of proteinase inhibitors in experimental anti-GBM glomerulopathy

Hruby¹,

Wendycz²,

Kopeć³

et al. 1999

Res. Exp. Med.

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We have previously documented amelioration of rat autologous anti-GBM nephritis with the antiproteolytic drugs epsilon-aminocaproic acid (EACA) and aprotinin, given from the day of induction or later in the course of disease. In the present study we investigated potential mechanisms of this effect by assessing interactions of the drugs with proteinase-dependent generation of superoxide anion in glomeruli, and their influence on both GBM degradation in vitro and activity of glomerular proteolytic enzymes. Release of O2- by enzymatically disrupted glomeruli, isolated from nephritic control or EACA/aprotinin-treated rats, was measured with the ferricytochrome reduction method and its activity was correlated with proteinuria and glomerular cellularity at the early phase of the disease. The hydroxyproline release assay was used to quantitate degradation of rat GBM in vitro by leukocyte proteinases stimulated by phorbol myristate acetate (PMA), in the presence or absence of EACA and aprotinin. Finally, the activities of elastase, cathepsins B and L, and plasmin, together with collagenase-like activity, were assessed fluorimetrically in homogenates of glomeruli isolated from control and antiproteolytic-drug-treated nephritic rats. EACA and aprotinin notably inhibited production of superoxide by nephritic glomeruli (by 47% and 66%, respectively), and this effect was not significantly correlated with proteinuria or glomerular hypercellularity at the early stage of disease. On the other hand, generation of O2- by glomeruli of untreated nephritic rats was notably correlated with total glomerular cell counts and numbers of macrophages infiltrating glomeruli. PMA-stimulated neutrophils and macrophages caused degradation of isolated rat GBM in vitro, markedly attenuated in the presence of EACA (P<0.0005) and, to a lesser extent, by addition of aprotinin (P<0.01). The activity of elastase was significantly reduced in glomeruli of nephritic rats treated with EACA or aprotinin (both P<0.001), while activities of remaining proteinases were not appreciably affected. The beneficial influence of proteinase inhibitors on rat anti-GBM disease may be due, at least in part, to abrogation of superoxide generation in nephritic glomeruli. EACA and aprotinin also have potential to interfere with digestion of GBM, and both these effects may be related to suppression of glomerular elastase.

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Direct photometric or fluorometric assay of proteinases using substrates containing 7-amino-4-trifluoromethylcoumarin

Cited by 94 publications

References 17 publications

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Dual‐Function Probe to Detect Protease Activity for Fluorescence Measurement and ¹⁹F MRI

Mechanism of antinephritic effect of proteinase inhibitors in experimental anti-GBM glomerulopathy

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Direct photometric or fluorometric assay of proteinases using substrates containing 7-amino-4-trifluoromethylcoumarin

Cited by 94 publications

References 17 publications

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Dual‐Function Probe to Detect Protease Activity for Fluorescence Measurement and 19F MRI

Mechanism of antinephritic effect of proteinase inhibitors in experimental anti-GBM glomerulopathy

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Dual‐Function Probe to Detect Protease Activity for Fluorescence Measurement and ¹⁹F MRI