Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins

Chagas, Jair Ribeiro; Juliano, Luiz; Prado, Eline S.

doi:10.1016/0003-2697(91)90558-b

Cited by 130 publications

(110 citation statements)

References 18 publications

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“…Hydrolysis of the intramolecularly fluorogenic quenched peptide substrates at 37 o C in 50 mM Tris-HCl, 2 mM CaCl 2 , pH 8.0, was monitored by measuring fluorescence at l em = 420 nm and l ex = 320 nm with a Hitachi F-2000 spectrofluorometer, as previously described (7). Standard hydrolysis conditions were strictly maintained for different substrates.…”

Section: Enzymatic Assaysmentioning

confidence: 99%

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

Fernandes

Anéas

Juliano

et al. 2000

Braz J Med Biol Res

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The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-AlaGln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a K m of 4.6 µM, a k cat of 1.73 s -1 , and a catalytic efficiency of 376 (mM s) -1 . Another good substrate for ZapA was peptide 6 (AbzArg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a K m of 13.6 µM, a k cat of 3.96 s -1 and a catalytic efficiency of 291 (mM s) -1 . The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P 1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P 2 .

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Section: Enzymatic Assaysmentioning

confidence: 99%

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

Fernandes

Anéas

Juliano

et al. 2000

Braz J Med Biol Res

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“…HOE-140 (D-Arg-[Hyp 3 -Thi 5 -D-Tic 7 -Oic 8 ]-bradykinin) (Hoechst, Germany). Phenylacetyl-FSR-EDDnp was obtained by peptide synthesis in solution using anhydride procedures and tert-butyloxycarbonyl-amino acids, puri®ed in silica gel and characterized by HPLC as previously described (Juliano & Juliano, 1985;Chagas et al, 1991). PKSI-527 was provided by Dr Yoshio Okada from the Faculty of Pharmacy Sciences, Kobe-Gakuin University, Kobe, Japan.…”

Section: Carrageenin-induced Peritonitismentioning

confidence: 99%

Evidence for activation of the tissue kallikrein‐kinin system in nociceptive transmission and inflammatory responses of mice using a specific enzyme inhibitor

Emim¹,

Souccar²,

Castro³

et al. 2000

British J Pharmacology

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1 The pharmacological activity of phenylacetyl-Phe-Ser-Arg-N-(2,4-dinitrophenyl)-ethylenediamine (TKI), a tissue kallikrein speci®c inhibitor, was assessed using models of nociception and in¯ammation in mice. 2 Injection of TKI (13.6 ± 136 mmol kg 71 , i.p. or 41 ± 410 mmol kg 71 , s.c.) produced a dose-related inhibition of the acetic acid-induced writhes (by 37 to 85% or 34 to 80%, respectively). The antinociceptive activity of TKI (41 mmol kg 71 , i.p.) was maximal after 30 min injection and lasted for 120 min. The e ect was unaltered by pretreatment with naloxone (8.2 mmol kg 71 , s.c.) or bilateral adrenalectomy. 3 TKI (41 and 136 mmol kg 71 , i.p.) produced a dose-related decrease of the late phase of formalininduced nociception by 79 and 98%, respectively. At 136 mmol kg 71 , i.p., TKI also shortened the duration of paw licking in the early phase by 69%. TKI (41 and 136 mmol kg 71 , i.p.) also reduced the capsaicin-induced nociceptive response (by 51 to 79%). 4 TKI (41 mmol kg 71 , i.p. or 410 mmol kg 71 , s.c.) reduced the oedematogenic response, from the second to the ®fth hour after carrageenin injection by 36 to 30% or by 47 to 39%, respectively. 5 Pretreatment with TKI (41 mmol kg 71 , i.p.) reduced the capsaicin-induced neurogenic in¯ammation in the mouse ear by 54%. 6 It is concluded that TKI presents antinociceptive and antiin¯ammatory activities mediated by inhibition of kinin formation by tissue kallikrein in mice. The results also indicate that the tissue kallikrein-dependent pathway contributes to kinin generation in nociceptive and in¯ammatory processes in mice.

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“…In contrast, intramolecularly quenched fluorogenic (IQF) peptide substrates, in which a fluorescent group (donor) is placed at one end of the substrate and a quencher group (acceptor) at the other end (Yaron et al, 1979), allow the design of substrates with amino acids on both sides of the scissile bond; such a substrate would be best accommodated in the enzyme's binding site. Several donor-acceptor pairs have been used for the characterization of peptidases: (a) o-aminobenzoic acid (Abz) as the donor with (4-N0,)Phe (Camel and Yaron, 1978), nitrobenzylamide (Hersh et al, 1983), 2,4-dinitrophenyl (Dnp) (Chagas et al, 1991), or (3-N02)Tyr (Meldal and Breddam, 1991) as the acceptor; (b) aminocoumarin and aminoquinolineone or nitroaromatic derivatives as the quencher (Tisljar et al, 1990); and (c) 5-[(2'-aminoethyl)amino]naphthalenesulfonic acid as the fluorescent group and 4-[(4'-(dimethylamino)phenyl)azo]benzoic acid as the quencher (Matayoshi et al, 1990) and tryptophan and the dansyl group as the acceptor (Ng and Auld, 1989). We have used peptides with Abz as the donor and Dnp as the acceptor because with this pair peptide hydrolysis results in a seven-to 100-fold increase in fluorescence as compared with the fluorescence of substrate and Dnp is a stable quencher that does not change its absorption spectrum with pH (Hirata et al, 1994).…”

Section: Peptide-amino-4-methylcoumarin (Amc) Substratesmentioning

confidence: 99%

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

Berman

Juliano

Devi

1999

Journal of Neurochemistry

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Abstract:The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, DnpArg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.

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Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins

Cited by 130 publications

References 18 publications

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

Evidence for activation of the tissue kallikrein‐kinin system in nociceptive transmission and inflammatory responses of mice using a specific enzyme inhibitor

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

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