Photometric or fluorometric assay of cathepsin B, L and H and papain using substrates with an aminotrifluoromethylcoumarin leaving group

doi:10.1016/0167-4838(91)90232-O

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1076, Issue 1, 8 January 1991, Pages 149-151

Biochimica et Biophy...

https://doi.org/10.1016/0167-4838(91)90232-O Get rights and content

Abstract

N-trifluoromethylcoumarinylamide derivatives of benzyloxycarbonyl-Arg-Arg, benzyloxycarbonyl-Phe-Arg and Arg are convenient chromogenic and fluorogenic substrates of cathepsin B, L and H, respectively. Benzyloxycarbonyl-Phe-Arg-N-trifluoromethylcoumarinylamide is also a highly sensitive substrate for papain.

Reference (14)

LahT.T. et al.
Biochim. Biophys. Acta
(1989)
SmithR.E. et al.
Thrombosis Res.
(1980)
CastilloM.J. et al.
Anal. Biochem.
(1979)
BrynesP.J. et al.
Anal. Biochem.
(1982)
KirschkeH. et al.
FEBS Lett.
(1984)
MoreauT. et al.
Biochem. Biophys. Res. Commun.
(1990)
BarrettA.J. et al.
Methods Enzymol.
(1981)

There are more references available in the full text version of this article.

Cited by (68)

Molecular probes for selective detection of cysteine cathepsins
2021, Organic and Biomolecular Chemistry
Cysteine cathepsins are proteases critical in physiopathological processes and show potential as targets or biomarkers for diseases and medical conditions. The 11 members of the cathepsin family are redundant in some cases but remarkably independent of others, demanding the development of both pan-cathepsin targeting tools as well as probes that are selective for specific cathepsins with little off-target activity. This review addresses the diverse design strategies that have been employed to accomplish this tailored selectivity among cysteine cathepsin targets and the imaging modalities incorporated. The power of these diverse tools is contextualized by briefly highlighting the nature of a few prominent cysteine cathepsins, their involvement in select diseases, and the application of cathepsin imaging probes in research spanning basic biochemical studies to clinical applications.
Screening of cathepsin B inhibitors in traditional Chinese medicine by capillary electrophoresis with immobilized enzyme microreactor
2019, Journal of Pharmaceutical and Biomedical Analysis
A simple and valid method for rapid screening of cathepsin B inhibitors from traditional Chinese medicines (TCMs) was established by the combination of immobilized enzyme microreactor (IMER) and capillary electrophoresis. Cathepsin B was immobilized on the inner surface of the capillary by glutaraldehyde method. The separation of substrate and product could be finished by baseline within 3 min. The activity of the immobilized cathepsin B remained approximately 90% after 50 runs. The quantification and statistical analysis of the product peak area was used to evaluate the catalytic activity of cathepsin B. The value of Michaelis-Menten constant of cathepsin B was 0.85 mM. The half-maximal inhibitory concentration (IC₅₀) of _L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) was measured as 36.08 nM, which indicated that the cathepsin B reactor was successfully developed and was feasible for inhibitorscreening. The raised method was then applied to discover the inhibitory potential of 17 standard compounds from traditional Chinese medicines. Five natural products, including kaempferol, rutaecarpine, evodiamine, theophylline, lycobetaine showed potential inhibition for cathepsin B. Additionally, molecular docking study was investigated for supporting the interaction between enzyme and inhibitors.
Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms
2017, International Journal for Parasitology
Citation Excerpt :
Cathepsin cysteine protease activity in supernatants of Giardia-spent medium, and in biofilm/Giardia-conditioned medium co-cultures, was measured via liberation of 7-aminomethylcoumarin (AMC) from fluorogenic substrates, using a Spectramax microplate reader (Molecular Devices Corp.). This method has been validated through cysteine protease-mediated proteolytic processing, and the change in reflective light units (RFUs), which can be used to calculate slope (Barrett, 1980; Tchoupe et al., 1991). Assays were performed at pH 7.2 in Cathepsin buffer (100 mM sodium acetate, 10 mM DTT, 0.1% Triton X-100, 1 mM EDTA, 0.5% DMSO – all from Sigma) in the presence/absence of broad-spectrum cysteine protease inhibitor E-64, and Cathepsin B inhibitor Ca-074Me, at concentrations of 1 μM, which was previously shown to have no detrimental effect on trophozoite viability (Rodriguez-Fuentes et al., 2006).
Giardia duodenalis is a prevalent cause of acute diarrheal disease worldwide. However, recent outbreaks in Italy and Norway have revealed a link between giardiasis and the subsequent development of chronic post-infectious irritable bowel syndrome. While the mechanisms underlying the causation of post-infectious irritable bowel syndrome remain obscure, recent findings suggest that alterations in gut microbiota communities are linked to the pathophysiology of irritable bowel syndrome. In the present study, we use a laboratory biofilm system to culture and enrich mucosal microbiota from human intestinal biopsies. Subsequently, we show that co-culture with Giardia induces disturbances in biofilm species composition and biofilm structure resulting in microbiota communities that are intrinsically dysbiotic – even after the clearance of Giardia. These microbiota abnormalities were mediated in part by secretory-excretory Giardia cysteine proteases. Using in vitro cell culture and germ-free murine infection models, we show that Giardia-induced disruptions of microbiota promote bacterial invasion, resulting in epithelial apoptosis, tight junctional disruption, and bacterial translocation across an intestinal epithelial barrier. Additionally, these dysbiotic microbiota communities resulted in increased activation of the Toll-like receptor 4 signalling pathway, and overproduction of the pro-inflammatory cytokine IL-1beta in humanized germ-free mice. Previous studies that have sought explanations and risk factors for the development of post-infectious irritable bowel syndrome have focused on features of enteropathogens and attributes of the infected host. We propose that polymicrobial interactions involving Giardia and gut microbiota may cause persistent dysbiosis, offering a new interpretation of the reasons why those afflicted with giardiasis are predisposed to gastrointestinal disorders post-infection.
Cloning and characterization of a basic cysteine-like protease (cathepsin L1) expressed in the gut of larval Diaprepes abbreviatus L. (Coleoptera: Curculionidae)
2015, Journal of Insect Physiology
Citation Excerpt :
Furthermore, no insect basic cathepsin L have been previously identified. Though Z-FR-AFC is an established cathepsin L substrate (Tchoupe et al., 1991), cathepsins B and H have affinities also for it. The general cathepsin inhibitor E-64 inhibits the activities of almost all cysteine proteases, including cathepsins B, H, and L (Barrett et al., 1982; Turk et al., 2012) but not cathepsin C (Rozman-Pungercar et al., 2003).
Diaprepes abbreviatus is an important pest that causes extensive damage to citrus in the USA. Analysis of an expressed sequence tag (EST) library from the digestive tract of larvae and adult D. abbreviatus identified cathepsins as major putative digestive enzymes. One class, sharing amino acid sequence identity with cathepsin L’s, was the most abundant in the EST dataset representing 14.4% and 3.6% of the total sequences in feeding larvae and adults, respectively. The predominant cathepsin (Da-CTSL1) among this class was further studied. Three dimensional modeling of the protein sequence showed that the mature Da-CTSL1 protein folds into an expected cathepsin L structure producing a substrate binding pocket with appropriate positioning of conserved amino acid residues. A full-length cDNA was obtained and the proCTSL1 encoding sequence was expressed in Rosetta™ Escherichia coli cells engineered to express tRNAs specific for eukaryotic codon usage. The Da-CTSL1 was expressed as a fusion protein with GST and His₆ tags and purified in the presence of 1% Triton X-100 by Ni-NTA affinity and size exclusion chromatography. Recombinant mature Da-CTSL1 (23 KDa) exhibits optimal activity at pH 8, rather than at acidic pH that was shown of all previously characterized cathepsins L. Substrate specificity supports the hypothesis that Da-CTSL1 is a unique basic cathepsin L and protease inhibitor studies also suggest unique activity, unlike other characterized acidic cathepsin Ls. This paper describes for the first time a prokaryotic expression system for the production of a functional eukaryotic cathepsin L1 from larval gut of D. abbreviatus.
Ultrasensitive internally quenched substrates of human cathepsin L
2014, Analytical Biochemistry
Internally quenched cathepsin L (Cat L) substrate ABZ-Bip-Arg-Ala-Gln-Tyr(3-NO₂)-NH₂ with high specificity constant (k_cat/K_M = 2.6 × 10⁷ M⁻¹ s⁻¹) was synthesized. The resultant compound displayed high selectivity over other members of the cathepsin family (B, S, X, V, C, K, H, F, D, and A). Activity of Cat L at picomolar (pM) concentrations was found using this substrate. Moreover, it was established that the presence of the selective Cat L inhibitor suppressed the proteolysis of the substrate to a non-detectable level. Incubation of the synthesized compound with a cell lysate of healthy and cancer cell lines indicated significant differences in Cat L activity. Based on the obtained results, it is proposed that this substrate could be used for selective monitoring of Cat L activity in biological systems.
Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures
2014, Analytical Biochemistry
This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp = pyroglutamyl; Xaa = Phe or Val; and Y = pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes.

View all citing articles on Scopus

View full text

Photometric or fluorometric assay of cathepsin B, L and H and papain using substrates with an aminotrifluoromethylcoumarin leaving group

Abstract

Biochim. Biophys. Acta

Thrombosis Res.

Anal. Biochem.

Anal. Biochem.

FEBS Lett.

Biochem. Biophys. Res. Commun.

Methods Enzymol.