A Microtiter Plate Assay for the Characterization of Serine Proteases by Their Esterase Activity

Whittaker, Robert G.; Manthey, Michael K.; Lebrocque, D.S.; Hayes, Patricia J.

doi:10.1006/abio.1994.1333

Cited by 28 publications

(15 citation statements)

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“…To confirm the presence of serine protease activities in trypsin and thrombin and to ensure the effective inhibition of protease activity by AEBSF, we used a microplate protease activity assay with a specific substrate for serine protease (39). In brief, 150 l substrate mix (6.3 mM Z-Ala-Arg-OMe, 6.3 mM bicine buffer, 0.068% phenol red, pH 9) was mixed with 40 l enzyme solution (serial dilutions of thrombin or trypsin in distilled water with 2 mM CaCl 2 and 1 mg/ml BSA) in the wells of 96-well tissue culture plates.…”

Section: Quantitation Of Serine Protease Activitymentioning

confidence: 99%

“…In a preliminary experiment, we used a microplate protease activity assay to determine the optimal conditions for inhibition of serine protease activity (39). Incubating 5 nM trypsin with a potent serine protease inhibitor, 2 mM AEBSF, for 30 min at 37°C abolished the serine protease activity.…”

Section: Blocking Protease Activity Of Trypsin Inhibits Trypsin-inducmentioning

confidence: 99%

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Trypsin Induces Activation and Inflammatory Mediator Release from Human Eosinophils Through Protease-Activated Receptor-2

Miike

McWilliam

Kita

2001

The Journal of Immunology

139

122

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Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50∼70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.

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Section: Quantitation Of Serine Protease Activitymentioning

confidence: 99%

Section: Blocking Protease Activity Of Trypsin Inhibits Trypsin-inducmentioning

confidence: 99%

Trypsin Induces Activation and Inflammatory Mediator Release from Human Eosinophils Through Protease-Activated Receptor-2

Miike

McWilliam

Kita

2001

The Journal of Immunology

139

122

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“…Serine protease activities in culture supernatants were measured using a previously developed microtitre plate esterase assay [18]. Briefly, synthetic substrates in the form benzyloxy carbonyl alanyl X methyl ester (Z‐Ala‐X‐Ome), where X was one of the 20 amino acids, were resuspended in DMSO to a final concentration of 20 mM.…”

Section: Methodsmentioning

confidence: 99%

Elevated levels of prostate‐specific antigen (PSA) in prostate cancer cells expressing mutant p53 is associated with tumor metastasis

Downing

Bumak

Nixdorf

et al. 2003

Molecular Carcinogenesis

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The underlying basis for rising levels of prostate-specific antigen (PSA) in prostate cancer is not fully understood, but attention has turned to the possibility that loss of normal p53 function might be directly involved. We have investigated the relationship between p53 function and PSA expression using in vitro and in vivo approaches. Three prostate cancer-derived p53 mutants (F134L, M237L, R273H) were introduced into LNCaP prostate cancer cells and stable transfectants established. Expression of mutant p53 was demonstrated by Western blot analysis, inactivation of wtp53 function, and a loss of p53-dependent responses to DNA damage induced by UV-irradiation and cisplatin. Levels of PSA mRNA and secreted protein were determined by RT-PCR and Western blotting, respectively. Serine protease activity was assessed using an esterase assay. In vivo effects of mutant p53 expression were examined after orthotopic implantation into prostates of nude mice. Expression of all p53 mutants was associated with elevated PSA mRNA and secreted PSA protein. In a representative line, mutant p53 was also associated with increased PSA protease-like activity compared with a control line expressing wildtype p53. Overall PSA levels, and PSA levels in serum from mice bearing tumors derived from cells expressing mutant p53, were increased compared with levels in mice bearing tumors derived from control cells. In addition, the tumors derived from cells with mutant p53 had increased vascularization and induced lymph node metastases. These data provide in vitro and in vivo support for the notion that p53 mutations directly contribute to increased levels of serum PSA, and are associated with more aggressive tumors.

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“…The catalytic mechanisms of serine proteases and the family of serine esterases to which PPT belongs are highly similar. These similarities are underscored by the observations that the serine proteases have been reported to have esterase activity (38,39) and that certain thioesterases have been reported to have protease activity (40). Thus, the observed correlation of lipofuscin induction and increases in endogenous or artificial serine protease inhibitors may be due to the inhibition of PPT, and it is possible that PPT also has protease activity.…”

Section: Maspgyrrll Aaallpwcca Awalghldpp Sppplviwhg Mgdsccnpms Mgsikmentioning

confidence: 99%

Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis.

Crews

Lane

Schreiber

1996

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

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A Microtiter Plate Assay for the Characterization of Serine Proteases by Their Esterase Activity

Cited by 28 publications

References 0 publications

Trypsin Induces Activation and Inflammatory Mediator Release from Human Eosinophils Through Protease-Activated Receptor-2

Trypsin Induces Activation and Inflammatory Mediator Release from Human Eosinophils Through Protease-Activated Receptor-2

Elevated levels of prostate‐specific antigen (PSA) in prostate cancer cells expressing mutant p53 is associated with tumor metastasis

Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis.

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