Intramolecularly quenched fluorogenic peptide substrates for human renin

Oliveira, Maria C. F. de; Hirata, Izaura Yoshico; Chagas, Jair Ribeiro; Boschcov, Paulo; Gomes, Roseli Aparecida da Silva; Figueiredo, Amintas F. S.; Juliano, Luiz

doi:10.1016/0003-2697(92)90040-e

Cited by 55 publications

(37 citation statements)

References 39 publications

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“…Hydrolysis was monitored at 11 sec intervals by fluorimetry in an M2e Microplate Reader (Molecular Devices Corporation, Sunnyvale, USA). The wavelength pair for excitation and emission was 370 nm/460 nm (Oliveira et al 1992). Enzyme activity is given by the enzyme initial rate obtained from kinetic measurements, where 1 enzyme unit (U) corresponds to an increase by 1 relative fluorescence unit (RFU) per sec.…”

Section: Cysteine Endopeptidase Activity Assaymentioning

confidence: 99%

Localization and function ofRhipicephalus (Boophilus) microplusvitellin-degrading cysteine endopeptidase

et al. 2010

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S U M M A R YThe tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.

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Section: Cysteine Endopeptidase Activity Assaymentioning

confidence: 99%

Localization and function ofRhipicephalus (Boophilus) microplusvitellin-degrading cysteine endopeptidase

et al. 2010

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“…based on HPLC-electrospray-tandem mass spectrometry, 7 uorogenic substrates 8-10 or FRET. [11][12][13] For uorogenic substrates, however, a uorophore must be placed next to the cleavage site, and FRET substrates are only quenched efficiently if the distance between the donor and acceptor is typically in the range of 2 to 6 nm.…”

Section: Introductionmentioning

confidence: 99%

Kinetic analysis of renin and its inhibitors by detecting double-labelled peptidic substrates with an immunoassay

Gorris

2013

Analyst

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The proteolytic activity of renin is a key element in the regulation of blood pressure and a main target for inhibitor design. Currently, the activity of renin and its inhibitors is mainly analyzed using radioimmunoassays or FRET-substrates, which both have their limitations. Here, a novel kinetic assay is presented that combines the advantages of a homogeneous proteolytic reaction and a robust heterogeneous detection in a sandwich immunoassay format. The proteolysis in solution is not influenced by surface interactions and yields accurate kinetic values, while the specific detection of the cleavage products on a microtiter plate strongly reduces interference by concomitant substances and allows for a self-referenced signal readout. A new enzyme kinetic scheme for the inhibition of renin has been developed and validated by using the model inhibitor pepstatin. This kinetic analysis is amenable to parallelization for large-scale inhibitor screening. Furthermore, it can be easily adapted to inhibitors of other medically important proteases.

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“…Peptides up to 20 residues can provide significant increases in florescence (de Souza et al 2000), allowing the measurement of the enzymatic activity on continuous base. The FRET peptides introduced by Chagas et al (1991) was a breakthrough in the study of proteases' specificity, and the synthesis of different Abzpeptidyl-EDDnp sequences provided the opportunity for us to study the activity of various endopeptidases such as human renin (Oliveira et al 1992), kallikreins (Chagas et al 1995, Del Nery et al 1995, Portaro et al 1997, Angelo et al 2006), cathepsin G (Réhault et al 1999, Korkmaz et al 2008), cathepsin D (Pimenta et al 2000, pro hormone convertase (Johanning et al 1998), lysosomal cathepsins (Portaro et al 2000, Alves et al 2003, Puzer et al 2004) and neprilysin (Medeiros et al 1997).…”

Section: Introductionmentioning

confidence: 99%

The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

Carmona

Juliano

2009

An. Acad. Bras. Ciênc.

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Proteolytic enzymes have a fundamental role in many biological processes and are associated with multiple pathological conditions. Therefore, targeting these enzymes may be important for a better understanding of their function and development of therapeutic inhibitors. Fluorescence Resonance Energy Transfer (FRET) peptides are convenient tools for the study of peptidases specificity as they allow monitoring of the reaction on a continuous basis, providing a rapid method for the determination of enzymatic activity. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that permits the measurement of the activity of nanomolar concentrations of the enzyme. The assays can be performed directly in a cuvette of the fluorimeter or adapted for determinations in a 96-well fluorescence plate reader. The synthesis of FRET peptides containing ortho-aminobenzoic acid (Abz) as fluorescent group and 2,4-dinitrophenyl (Dnp) or N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as quencher was optimized by our group and became an important line of research at the Department of Biophysics of the Federal University of São Paulo. Recently, Abz/Dnp FRET peptide libraries were developed allowing high-throughput screening of peptidases substrate specificity. This review presents the consolidation of our research activities undertaken between 1993 and 2008 on the synthesis of peptides and study of peptidases specificities.

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Intramolecularly quenched fluorogenic peptide substrates for human renin

Cited by 55 publications

References 39 publications

Localization and function ofRhipicephalus (Boophilus) microplusvitellin-degrading cysteine endopeptidase

Localization and function ofRhipicephalus (Boophilus) microplusvitellin-degrading cysteine endopeptidase

Kinetic analysis of renin and its inhibitors by detecting double-labelled peptidic substrates with an immunoassay

The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

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