Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization

Background: Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.Results: A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-alpha-benzoyl-(DL)-arginine-p-nitroanilide (BApNA) as substrate. in addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50 degrees C, respectively. After incubation at 50 degrees C for 30 min the enzyme lost only 20% of its activity. K-m, k(cat), and k(cat)/K-m values using BApNA as substrate were 0.689 mM, 6.9 s(-1), and 10 s(-1) mM(-1), respectively. High inhibition of trypsin activity was observed after incubation with Cd2+, Al3+, Zn2+, Cu2+, Pb2+, and Hg2+ at 1 mM, revealing high sensitivity of the enzyme to metal ions.Conclusions: Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.Financiadora de Estudos e Projetos (FINEP/RECARCINE)Petroleo do Brasil S/A (PETROBRAS)Secretaria Especial de Aquicultura e Pesca (SEAP/PR)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE)Univ Fed Pernambuco, Lab Enzimol LABENZ, Dept Bioquim CCB, BR-50670910 Recife, PE, BrazilUniv Fed Pernambuco, LIKA, BR-50670910 Recife, PE, BrazilUniversidade Federal de São Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniv Fed Pernambuco, Lab Glicoprot, Dept Bioquim CCB, BR-50670910 Recife, PE, BrazilUniversidade Federal de São Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 São Paulo, BrazilWeb of Scienc

CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.-Berezniuk, I., Sironi, J., Callaway, M. B., Castro, L. M., Hirata, I. Y., Ferro, E. S., Fricker, L. D. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813-1823 (2010). www.fasebj.orgU.S. National Institutes of Health (NIH)National Institutes of Health (NIH)[DK-51271]National Institutes of Health (NIH)[DA-04494]U.S. National Institutes of Health (NIH)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[04/04933-2]FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[04/14846-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Financiadora de Estudos e Projetos (FINEP)[A-03/134]Financiadora de Estudos e Projetos (FINEP)CNPq Conselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade de Campinas (UNICAMP), Campinas, SP, BrazilUniversidade Estadual de Campinas (UNICAMP

Controlled peptide solvation in portion-mixing libraries of FRET peptides: Improved specificity determination for dengue 2 virus NS2B-NS3 protease and human cathepsin S

The solubility of peptides in aqueous buffers used for the enzyme assays is a common limitation for all peptide libraries. in principle, the more water-soluble peptides are, the more susceptible they will be to peptidase hydrolysis. We have demonstrated that this bias can be circumvented in a portion-mixing fluorescence resonance energy transfer (FRET) peptide library by introducing k (lysine in the D-form) in both termini of the peptides. This more solvated library and another one without the k were assayed using trypsin and chymotrypsin as standard peptidases with high selectivity for R and K and for hydrophobic F and Y, respectively. Significantly improved consistency of the information on substrate profiles was obtained from the solvated library. the influence of improved solvation on substrate specificity determination was successfully demonstrated by the difference in specificity observed between the two libraries employing the human cathepsin S (accepts acidic, basic, or neutral amino acids at P(1) position) and Dengue 2 virus NS2B-NS3 protease (high specificity to the pair of basic amino acids K-R, R-R, or Q-R/K at P(2)-P(1) positions). in conclusion, hydration of the peptides has a major influence on protease processing, and this bias can be reduced in bound peptide libraries, improving reliability.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilCtr Gamle Carlsberg, SPOCC, Carlsberg Lab, DK-2500 Copenhagen, DenmarkUniv British Columbia, Dept Dent, Vancouver, BC V6T 1Z3, CanadaUniv British Columbia, UBC Ctr Blood Res, Vancouver, BC V6T 1Z3, CanadaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Purification and partial characterization of human neutrophil elastase inhibitors from the marine snail Cenchritis muricatus (Mollusca)

Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 C for 30min. From this extract, three human neutrophil elastase inhibitors (designated CmPI-I, CmPI-II and CmPI-III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI-I and CmPI-II was confirmed, while CmPI-III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0kDa. in contrast, MALDI-TOF mass spectrometry of CmPI-I and CmPI-II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI-I (6 amino acids) and CmPI-II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI-II. Both inhibitors, CmPI-I and CmPI-II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (K;) values of 54.2 and 1.6nM, respectively. in addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI-I and CmPI-II are the first human neutrophil elastase inhibitors described in a mollusk. (c) 2006 Elsevier Inc. All rights reserved.Univ La Habana, Fac Biol, Ctr Estudio Prot, Havana, CubaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilWeb of Scienc

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum)

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex (R) G-75 and p-aminobenzamidine-agarose affinity chromatography. the enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3 h at 60 degrees C. the enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry. (C) 2010 Elsevier Inc. All rights reserved.Ministry of Fisheries and AquacultureCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FINEPFACEPEPETROBRASUniv Fed Pernambuco, LABENZ, Dept Bioquim CCB, BR-50670910 Recife, PE, BrazilUniv Fed Pernambuco, LIKA, BR-50670910 Recife, PE, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Plasminogen hydrolysis by cathepsin S and identification of derived peptides as selective substrate for cathepsin V and cathepsin L inhibitor

Plasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively We have recently reported that cathepsin V. but not cathepsins L. B, and K. can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. in contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. the peptide Abz-VLFEKKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cathepsin V at the Phe-Glu bond. is a selective substrate for the enzyme when compared with cathepsins B. L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. the demonstrated importance of the S-3'-P-3' interaction indicates the significance of the extended subsites for enzyme specificity and affinity.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Canada Research Chair AwardUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv Fed Triangulo Mineiro, Dept Biol Sci, BR-38025015 Uberaba, MG, BrazilUniv British Columbia, Dept Dent, Vancouver, BC V6T 1Z3, CanadaUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc