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ABSTRACT: Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.
Protein and Peptide Letters 11/2011; 19(4):474-7. · 1.94 Impact Factor
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ABSTRACT: Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas' disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 μg/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7·6-fold), heart (3-fold) and small intestine (3·6-fold). Moreover, an intense inflammatory response and increment of CD4+ T cells (1·7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4+CD25+FoxP3+ T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas' disease.
Parasitology 11/2010; 138(4):481-92. · 2.96 Impact Factor
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ABSTRACT: Insect chymotrypsins are distinctively sensitive to plant protein inhibitors, suggesting that they differ in subsite architecture and hence in substrate specificities. Purified digestive chymotrypsins from insects of three different orders were assayed with internally quenched fluorescent oligopeptides with three different amino acids at P1 (Tyr, Phe, and Leu) and 13 amino acid replacements in positions P1', P2, and P3. The binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T)++, determined from k(cat)/K(m) values) were calculated. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results showed that except for S1, the other subsites (S2, S3, and S1') vary among chymotrypsins. This result contrasts with insect trypsin data that revealed a trend along evolution, putatively associated with resistance to plant inhibitors. In spite of those differences, the data suggested that in lepidopteran chymotrypsins S2 and S1' bind the substrate ground state, whereas only S1' binds the transition state, supporting aspects of the present accepted mechanism of catalysis.
Insect Biochemistry and Molecular Biology 07/2008; 38(6):628-33. · 3.25 Impact Factor
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ABSTRACT: An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. The kinetic parameter K(m) for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. The specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. In conclusion, we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture.
Analytical Biochemistry 05/2007; 363(2):255-62. · 3.00 Impact Factor
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I E Gouvea,
M A Izidoro,
W A S Judice,
M H S Cezari,
G Caliendo,
V Santagada,
C N D dos Santos,
M H Queiroz, M A Juliano,
P R Young,
D P Fairlie,
L Juliano
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ABSTRACT: A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.
Archives of Biochemistry and Biophysics 02/2007; 457(2):187-96. · 2.93 Impact Factor
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ABSTRACT: S100A9 protein induces anti-nociception in rodents, in different experimental models of inflammatory pain. Herein, we investigated the effects of a fragment of the C-terminus of S100A9 (mS100A9p), on the hyperalgesia induced by serine proteases, through the activation of protease-activated receptor-2 (PAR2).
Mechanical and thermal hyperalgesia induced by PAR2 agonists (SLIGRL-NH2 and trypsin) was measured in rats submitted to the paw pressure or plantar tests, and Egr-1 expression was determined by immunohistochemistry in rat spinal cord dorsal horn. Calcium flux in human embryonic kidney cells (HEK), which naturally express PAR2, in Kirsten virus-transformed kidney cells, transfected (KNRK-PAR2) or not (KNRK) with PAR2, and in mouse dorsal root ganglia neurons (DRG) was measured by fluorimetric methods.
mS100A9p inhibited mechanical hyperalgesia induced by trypsin, without modifying its enzymatic activity. Mechanical and thermal hyperalgesia induced by SLIGRL-NH2 were inhibited by mS100A9p. SLIGRL-NH2 enhanced Egr-1 expression, a marker of nociceptor activation, and this effect was inhibited by concomitant treatment with mS100A9p. mS100A9p inhibited calcium mobilization in DRG neurons in response to the PAR2 agonists trypsin and SLIGRL-NH2, but also in response to capsaicin and bradykinin, suggesting a direct effect of mS100A9 on sensory neurons. No effect on the calcium flux induced by trypsin or SLIGRL in HEK cells or KNRK-PAR2 cells was observed.
These data demonstrate that mS100A9p interferes with mechanisms involved in nociception and hyperalgesia and modulates, possibly directly on sensory neurons, the PAR2-induced nociceptive signal.
British Journal of Pharmacology 11/2006; 149(4):374-84. · 4.41 Impact Factor
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ABSTRACT: Trypsins have high sequence similarity, although the responses of insect trypsins to chemical and natural inhibitors suggest they differ in specificities. Purified digestive trypsins from insects of four different orders were assayed with internally quenched fluorescent oligopeptides with two different amino acids at P1 (Arg/Lys) and 15 amino acid replacements in positions P1', P2', P2, and P3. The binding energy (deltaG(s), calculated from Km values) and the activation energy (deltaG(T)(double dagger), determined from kcat/Km values) were calculated. Dictyoptera, Coleoptera and Diptera trypsins hydrolyze peptides with Arg at P1 at least 3 times more efficiently than peptides with Lys at P1, whereas Lepidoptera trypsins have no preference between Arg and Lys at that position. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results suggested that insect trypsin subsites become progressively more hydrophobic along evolution. Apparently, this is an adaptation to resist plant protein inhibitors, which usually have polar residues at their reactive sites. Results also suggested that, at least in lepidopteran trypsins, S3, S2, S1', and S2' significantly bind the substrate ground state, whereas in the transition state only S1' and S2' do that, supporting aspects of the presently accepted mechanism of trypsin catalysis. Homology modeling showed differences among those trypsins that may account for the varied kinetic properties.
Insect Biochemistry and Molecular Biology 03/2006; 36(2):130-40. · 3.25 Impact Factor
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ABSTRACT: In the present study, the effect of a synthetic peptide (H(92)-G(102)) identical to the C-terminus of murine S100A9 (mS100A9p) was investigated on adherent peritoneal cell function.
For in vitro assays, peritoneal cells were obtained from the abdominal cavity of mice and incubated, with the different concentrations of mS100A9p, for 1 h, and then their spreading and phagocytosis activities were evaluated. For ex-vivo assays, cells obtained from animals treated for 1 h with the peptide were submitted to the mannose-receptor phagocytosis assay. Shorter homologue peptides to the C-terminus of mS100A9p were also evaluated on in vitro phagocytosis assays of Candida albicans particles.
mS100A9p reduced both the spreading index and phagocytic activity, in vitro and ex-vivo, independent of the receptor evaluated. The homologue peptide corresponding to the H(92)-E(97) region of mS100A9p, the zinc-binding motif, was responsible for such an effect.
These results suggest a modulator effect of the C-terminus of S100A9 protein on the function of adherent peritoneal cells.
Inflammation Research 06/2005; 54(5):204-10. · 2.11 Impact Factor
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ABSTRACT: A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambda ex = 320 nm and lambda em = 420 nm) at 37 degrees C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 microM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 microM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 microM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.
Brazilian Journal of Medical and Biological Research 06/2005; 38(6):861-8. · 1.13 Impact Factor
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ABSTRACT: In this study, we compared the properties of a serine endopeptidase H1 (SH1) and a serine thiol endopeptidase (STH2) purified from human urine by DEAE-cellulose followed by a Bio Gel A0.5 m or Sepharose Mercurial chromatographs. These enzymes differ in their action upon different hormone peptides. We used fluorogenic substrates to further characterize the enzyme. The substrate specificity of urinary SH1 was studied using different internally quenched fluorescent peptides, and AbzFGQEDDnp was hydrolyzed by SH1. Other enzymes present in urine, such as serine endopeptidase H2, prolyl endopeptidase, neutral endopeptidase like and angiotensin-I converting enzyme, were not able to hydrolyze this substrate. SH1 is 100% inhibited by PMSF and resistant to EDTA, OPA, thiorphan, E64, pOHMB and phosphoramidon. Endopeptidase STH2 is completely inhibited by PMSF, E64 and pOHMB. Enzyme SH1 hydrolyzes the peptide bound F5-S6 at bradykinin (BK: RPPGFSPFR) molecule and R-Q at AbzBKQEDDnp. When studying enzyme STH2, the cleavage sites determined to the related substrates were F5-S6 using BK as substrate and F-R using AbzBKQEDDnp. The kilometers value obtained for AbzBKQEDDnp and AbzFGQEDDnp were 1.18 and 0.007 uM, respectively. Kininases from kidney and urine can hydrolyze peptide bounds from components of the kallikrein-kinin system, the angiotensin-renin system and the neuropeptides system, straight contributing in kidney homeostasis. SH1 was located at the distal tubule [Casarini et al., 1999a, Am. J. Physiol. 277, F66] and can have an important function in the control of kinin found in this portion, since is known that all components of the kallikrein-kinin system were found in this portion. The physiological role of SHT2 could be related to the inter-relation between the kallikrein-kinin system and neuropeptides in the control of the water electrolyte balance [Braz. J. Med. Biol. Res. 25 (3) (1992) 219].
The International Journal of Biochemistry & Cell Biology 11/2004; 36(10):1933-44. · 4.63 Impact Factor
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ABSTRACT: Many plant proteinase inhibitors have lysine at the P1 position, presumably to avoid hydrolysis by insect trypsins. Lepidopteran trypsins appear to have adapted to resist proteinase inhibitors through increased inhibitor hydrolysis and decreased binding to inhibitor hydrophilic reactive sites. Lepidopteran digestive trypsins prefer lysine at the P1 position and have substrate binding subsites more hydrophobic than trypsins from insects in other orders. All available sequences of sensitive and inhibitor-insensitive insect trypsins were aligned with porcine trypsin, for which interactions with Kunitz and Bowman-Birk inhibitor are known. After discounting conserved positions and positions not typical of sensitive or insensitive trypsins, the following residues were considered important to insect trypsin-PI interactions (chymotrypsin numbering): 60, 94, 97, 98, 99, 188, 190, 213, 215, 217, 219, 228. These residues support the Neighbor Joining analysis tree branches separating sensitive and insensitive trypsin sequences. Primary sequences interacting with PIs are around the active site, with some forming part of the S1 (188, 217, 219 and 228) or S4 (99, 215) pockets.
Archives of Insect Biochemistry and Physiology 04/2004; 55(3):140-52. · 1.36 Impact Factor
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ABSTRACT: Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambda exc = 320 nm and lambda em = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.
Brazilian Journal of Medical and Biological Research 09/2003; 36(8):1047-53. · 1.13 Impact Factor
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ABSTRACT: Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.
Journal of Protein Chemistry 09/2003; 22(6):533-41.
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ABSTRACT: The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.
Current Medicinal Chemistry 07/2003; 10(13):1085-93. · 4.86 Impact Factor
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ABSTRACT: Enzymes enhance chemical reaction rates by lowering the activation energy, the energy barrier of the reaction leading to products. This occurs because enzymes bind the high-energy intermediate of the reaction (the transition state) more strongly than the substrate. We studied details of this process by determining the substrate binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T), determined from k(cat)/K(m) values) for the trypsin-catalyzed hydrolysis of oligopeptides. Plots of DeltaG(T) versus DeltaG(s) for oligopeptides with 15 amino acid replacements at each of the positions P(1)', P(1), and P(2) were straight lines, as predicted by a derived equation that relates DeltaG(T) and DeltaG(s). The data led to the conclusion that the trypsin active site has subsites that bind moieties of substrate and of transition state in characteristic ratios, whichever substrate is used. This was unexpected and means that each subsite characteristically favors substrate binding or catalysis.
Biochemical and Biophysical Research Communications 02/2002; 290(1):494-7. · 2.48 Impact Factor
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ABSTRACT: The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.
Journal of Biological Chemistry 01/2002; 276(52):49400-9. · 4.77 Impact Factor
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ABSTRACT: The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2' subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH-activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k-1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci. 9, 1589-1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E-1, which are significantly higher for cruzipain. The corresponding DeltaS1 and DeltaS-1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain.
European Journal of Biochemistry 12/2001; 268(24):6578-86. · 3.58 Impact Factor
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ABSTRACT: We have explored the specificity of the S(2) subsite of recombinant cysteine proteinases from Leishmania mexicana (CPB2.8 Delta CTE) and from Trypanosoma cruzi (cruzain) employing a series of fluorogenic substrates based on the peptide Bz-F-R-MCA, in which Bz is the benzoyl group and the Phe residue has been substituted for by Arg, His and non-natural basic amino acids that combine a basic group with an aromatic or hydrophobic group at the side chain: 4-aminomethyl-phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). Bz-F-R-MCA was hydrolyzed well by CPB2.8 Delta CTE and cruzain, but all the substitutions of Phe resulted in less susceptible substrates for the two enzymes. CPB2.8 Delta CTE has a restricted specificity to hydrophobic side chains as with cathepsin L. However, the peptides with the residues Amf and Ama presented higher affinity to CPB2.8 Delta CTE, and the latter was an inhibitor of the enzyme. Although, cruzain accepts basic as well as hydrophobic residues at the S(2) subsite, it is more restrictive than cathepsin B and no inhibitor was found amongst the examined peptides.
Molecular and Biochemical Parasitology 11/2001; 117(2):137-43. · 2.55 Impact Factor
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ABSTRACT: We have isolated and purified two cysteine proteinases of molecular weights 25 and 26 kDa, secreted by Fasciola hepatica adult worm. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L-like enzymes, isolated from the same source, reported as CL1 and CL2. Radioimmunoassay experiments have shown that these CL1- (25 kDa) and CL2-like (26 kDa) cysteine proteinases mediated kinin release from high molecular weight kininogen (HMWK). Lys-bradykinin (KRPPGFSPFR) was characterized as the kinin released from a synthetic fragment of HMWK from Leu373 to Ile393 (Abz-LGMISLMKRPPGFSPFRSSRI-NH2) labeled with the fluorescent group Abz (ortho-aminobenzoic acid). We examined the activity of CL1- and CL2-like on internally quenched fluorescent peptides containing HMWK sequences, in which Met379-Lys380 or Arg389-Ser390 bonds were present in the middle of the molecules. These peptides were flanked by the fluorescent donor-acceptor pair Abz and EDDnp (N-[2,4-dinitrophenyl] ethylenediamine). Peptidyl-methylcoumarin amides (MCA) were used to study the substrate specificity requirements. The enzymes presented significantly lower Km values at pH 8.0. The inverse was observed with the kcat values, which were higher at pH 5.0.
Molecular and Biochemical Parasitology 10/2001; 116(2):109-15. · 2.55 Impact Factor
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ABSTRACT: Micro-scale (sub-pmol) isolation and sequence determination of three peptides from the venom of the solitary spider wasp Cyphononyx dorsalis is described. We isolated two novel peptides Cd-125 and Cd-146 and a known peptide Thr(6)-bradykinin from only two venom sacs of solitary spider wasp Cyphononyx dorsalis without bioassay-guided fractionation, but instead guided by MALDI-TOF MS. The MALDI-TOF MS analysis of each fraction showed the purity and molecular weight of the components, which led to the isolation of the peptides virtually without loss of sample amount. The sequences of the novel peptides Cd-125 (Asp-Thr-Ala-Arg-Leu-Lys-Trp-His) and Cd-146 (Ser-Glu-Thr-Gly-Asn-Thr-Val-Thr-Val-Lys-Gly-Phe-Ser-Pro-Leu-Arg) were determined by Edman degradation together with mass spectrometry, and finally corroborated by solid-phase synthesis. The known peptide Thr(6)-bradykinin (Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) was identified by comparison with the synthetic authentic specimen. This is the first example for any kinins to be found in Pompilidae wasp venoms. The procedure reported here can be applicable to studies on many other components of solitary wasp venoms with limited sample availability.
Toxicon 09/2001; 39(8):1257-60. · 2.51 Impact Factor
