34 results on '"Chagas, J R"'
Search Results
2. Design of inhibitors for tissue kallikreins based on their extended binding site specificities
- Author
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Juliano, L., primary, Portaro, F. C. V., additional, Chagas, J. R., additional, Hirata, I. Y., additional, Juliano, M. A., additional, Bizeto, L., additional, Antunes, E., additional, de Nucci, G., additional, and Prado, E. S., additional
- Published
- 1995
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3. Determinants of Tissue Kallikrein Cleavage Specificity in the Limited Proteolysis of Kininogens
- Author
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Prado, E. S., primary, Chao, J., additional, Chagas, J. R., additional, Juliano, M. A., additional, and Juliano, L., additional
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- 1992
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4. Intramolecularly quenched fluorogenic peptide substrates for kallikreins and human renin
- Author
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Juliano, L., primary, Oliveira, M. C. F., additional, Chagas, J. R., additional, and Prado, E. S., additional
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- 1991
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5. Characterization of kininogenase activity of an acidic proteinase isolated from human kidney
- Author
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Juliano, L, Chagas, J R, Hial, V, and Gomes, R AS
- Published
- 1997
6. Purification and characterization of an alkaline serine endopeptidase from a feather-degradingXanthomonas maltophiliastrain
- Author
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De Toni, C H, primary, Richter, M F, additional, Chagas, J R, additional, Henriques, J AP, additional, and Termignoni, C, additional
- Published
- 2002
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7. Characterization of kininogenase activity of an acidic proteinase isolated from human kidney
- Author
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Gomes, RAS, primary, Juliano, L, additional, Chagas, J R, additional, and Hial, V, additional
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- 1997
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8. Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments
- Author
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Del Nery, E, primary, Chagas, J R, additional, Juliano, M A, additional, Prado, E S, additional, and Juliano, L, additional
- Published
- 1995
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9. Determinants of the unusual cleavage specificity of lysyl-bradykinin-releasing kallikreins
- Author
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Chagas, J R, primary, Portaro, F C V, additional, Hirata, I Y, additional, Almeida, P C, additional, Juliano, M A, additional, Juliano, L, additional, and Prado, E S, additional
- Published
- 1995
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10. The anti-leishmanial activity of dipeptide esters onLeishmania amazonensisamastigotes
- Author
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Ramazeilles, C., primary, Juliano, L., additional, Chagas, J. R., additional, and Rabinovitch, M., additional
- Published
- 1990
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11. Purification and characterization of an alkaline serine endopeptidase from a feather-degrading Xanthomonas maltophilia strain.
- Author
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De Toni, C H, Richter, M F, Chagas, J R, Henriques, J AP, and Termignoni, C
- Subjects
ENDOPEPTIDASES ,XANTHOMONAS ,KERATIN ,ENZYME inhibitors ,CAPILLARY electrophoresis - Abstract
A keratinolytic Xanthomonas maltophilia strain (POA-1), cultured on feather meal broth, using keratin as its sole source of carbon and nitrogen, secretes several extracellular peptidases. The major serine peptidase was purified to homogeneity by a five-step procedure. Its purity was evaluated by capillary zone electrophoresis. This enzyme has a molecular mass of 36 kDa, an optimum pH of 9.0, and an optimum temperature of 60°C. The inhibitory profile using protease inhibitors shows that this enzyme is a serine endopeptidase. Besides keratin, the enzyme is active upon the substrates azokeratin, azocasein, and the following fluorogenic peptide substrates: Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Gln-EDDnp, Abz-Lys-Leu-Cys(SBzl)-Gly-Pro-Lys-Gln-EDDnp, and Abz-Lys-Pro-Cys(SBzl)-Phe-Ser-Lys-Gln-EDDnp.Key words: serine endopeptidase, Xanthomonas maltophilia, keratinase, alkaline endopeptidase.Lorsque cultivé dans un bouillon à base de farine de plumes et qu'elle utilise la kératine comme source unique de carbone et d'azote, la souche kératinolytique de Xanthomonas maltophilia (POA-1) sécrète quelques peptidases extracellulaires. La principale enzyme, une sérine peptidase, a été purifiée à l'homogénéité selon un processus en cinq étapes. La pureté de l'enzyme a été évaluée par électrophorèse basée sur les zones observées en capillaires. Cette enzyme a une masse moléculaire de 36 kDa, un pH optimal de 9,0 et une température optimale de 60°C. En utilisant des inhibiteurs des protéases, le profil d'inhibition a révélé que cette enzyme était une sérine endopeptidase. Cette enzyme s'est montrée active sur d'autres substrats que la kératine comme l'azokératinine, l'azocaséine et les substrats peptidiques fluorogènes suivants : Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Gln-EDDnp, Abz-Lys-Leu-Cys(SBzl)-Gly-Pro-Lys-Gln-EDDnp et Abz-Lys-Pro-Cys(SBzl)-Phe-Ser-Lys-Gln-EDDnp.Mots clés : sérine endopeptidase, Xanthomonas maltophilia, kératinase, endoseptidase alcaline.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]
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- 2002
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12. Hydrolysis by cathepsin B of fluorescent peptides derived from human prorenin.
- Author
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Almeida, Paulo Cezar, Oliveira, Vitor, Chagas, Jair Ribeiro, Meldal, Morten, Juliano, Maria Aparecida, Juliano, Luiz, Almeida, P C, Oliveira, V, Chagas, J R, Meldal, M, Juliano, M A, and Juliano, L
- Published
- 2000
13. Cysteine proteinase activity regulation. A possible role of heparin and heparin-like glycosaminoglycans.
- Author
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Almeida, P C, Nantes, I L, Rizzi, C C, Júdice, W A, Chagas, J R, Juliano, L, Nader, H B, and Tersariol, I L
- Abstract
Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the alpha-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced alpha-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce alpha-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.
- Published
- 1999
14. A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi
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Chagas, J. R., Authie, E., Serveau, C., Lalmanach, G., Juliano, L., and Gauthier, F.
- Published
- 1997
- Full Text
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15. Inhibition of trypanosomal cysteine proteinases by their propeptides.
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Lalmanach, G, Lecaille, F, Chagas, J R, Authié, E, Scharfstein, J, Juliano, M A, and Gauthier, F
- Abstract
The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in Ki value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.
- Published
- 1998
16. The anti-leishmanial activity of dipeptide esters on Leishmania amazonensis amastigotes.
- Author
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Ramazeilles, C., Juliano, L., Chagas, J. R., and Rabinovitch, M.
- Published
- 1990
- Full Text
- View/download PDF
17. Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom.
- Author
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Higuchi DA, Barbosa CM, Bincoletto C, Chagas JR, Magalhaes A, Richardson M, Sanchez EF, Pesquero JB, Araujo RC, and Pesquero JL
- Subjects
- Amino Acid Sequence, Animals, Anticoagulants pharmacology, Blood Coagulation drug effects, Calcium metabolism, Carcinoma, Ehrlich Tumor chemically induced, Carcinoma, Ehrlich Tumor enzymology, Carcinoma, Ehrlich Tumor pathology, Cell Survival drug effects, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Enzyme-Linked Immunosorbent Assay, Hemoglobins metabolism, Humans, Isoenzymes chemistry, Isoenzymes isolation & purification, Isoenzymes metabolism, K562 Cells, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Molecular Weight, Phospholipases A chemistry, Phospholipases A isolation & purification, Platelet Aggregation drug effects, Sequence Analysis, Protein, Sequence Homology, Amino Acid, Snake Venoms pharmacology, Bothrops metabolism, Phospholipases A metabolism, Snake Venoms enzymology
- Abstract
Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.
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- 2007
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18. High expression of human carboxypeptidase M in Pichia pastoris: purification and partial characterization.
- Author
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Craveiro RB, Ramalho JD, Chagas JR, Wang PH, Casarini D, Pesquero JL, Araújo RC, and Pesquero JB
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- Chromatography, Ion Exchange, GPI-Linked Proteins, Humans, Metalloendopeptidases genetics, Pichia genetics, Reverse Transcriptase Polymerase Chain Reaction, Metalloendopeptidases isolation & purification, Pichia enzymology
- Abstract
Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.
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- 2006
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19. Proteinase activity regulation by glycosaminoglycans.
- Author
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Tersariol IL, Pimenta DC, Chagas JR, and Almeida PC
- Subjects
- Animals, Cysteine Endopeptidases metabolism, Glycosaminoglycans metabolism, Heparin physiology, Humans, Matrix Metalloproteinases metabolism, Serine Endopeptidases metabolism, Serine Proteinase Inhibitors metabolism, Tissue Inhibitor of Metalloproteinases metabolism, Endopeptidases metabolism, Glycosaminoglycans physiology
- Abstract
There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.
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- 2002
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20. Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruzi.
- Author
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Judice WA, Cezari MH, Lima AP, Scharfstein J, Chagas JR, Tersariol IL, Juliano MA, and Juliano L
- Subjects
- Amino Acid Sequence, Animals, Enzyme Activation, Enzyme Stability, Hot Temperature, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Oligopeptides metabolism, Protein Denaturation, Recombinant Proteins metabolism, Sodium Chloride, Substrate Specificity, Cysteine Endopeptidases metabolism, Protozoan Proteins metabolism, Trypanosoma cruzi enzymology
- 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.
- Published
- 2001
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21. Cathepsin B activity regulation. Heparin-like glycosaminogylcans protect human cathepsin B from alkaline pH-induced inactivation.
- Author
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Almeida PC, Nantes IL, Chagas JR, Rizzi CC, Faljoni-Alario A, Carmona E, Juliano L, Nader HB, and Tersariol IL
- Subjects
- Amino Acid Substitution, Cathepsin B isolation & purification, Chondroitin Sulfates metabolism, Chondroitin Sulfates pharmacology, Chromatography, Affinity, Circular Dichroism, Cloning, Molecular, Dermatan Sulfate metabolism, Dermatan Sulfate pharmacology, Dextran Sulfate metabolism, Dextran Sulfate pharmacology, Enzyme Stability, Glycosaminoglycans pharmacology, Heparin pharmacology, Heparitin Sulfate pharmacology, Humans, Hydrogen-Ion Concentration, Kinetics, Liver enzymology, Protein Denaturation drug effects, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Cathepsin B chemistry, Cathepsin B metabolism, Glycosaminoglycans metabolism, Heparin metabolism, Heparitin Sulfate metabolism
- Abstract
It has been shown that lysosomal cysteine proteinases, specially cathepsin B, has been implicated in a variety of diseases involving tissue remodeling states, such as inflammation, parasite infection, and tumor metastasis, by degradation of extracellular matrix components. Recently, we have shown that heparin and heparan sulfate bind to papain specifically; this interaction induces an increase of its alpha-helix content and stabilizes the enzyme structure even at alkaline pH (Almeida, P. C., Nantes, I. L., Rizzi, C. C. A., Júdice, W. A. S., Chagas, J. R., Juliano, L., Nader, H. B., and Tersariol, I. L. S. (1999) J. Biol. Chem. 274, 30433-30438). In the present work, a combination of circular dichroism analysis, affinity chromatography, cathepsin B mutants, and fluorogenic substrate assays were used to characterize the interaction of human cathepsin B with glycosaminoglycans. The nature of the cathepsin B-glycosaminoglycans interaction was sensitive to the charge and type of polysaccharide. Like papain, heparin and heparan sulfate bind cathepsin B specifically, and this interaction reduces the loss of cathepsin B alpha-helix content at alkaline pH. Our data show that the coupling of cathepsin B with heparin or heparan sulfate can potentiate the endopeptidase activity of the cathepsin B, increasing 5-fold the half-life (t(12)) of the enzyme at alkaline pH. Most of these effects are related to the interaction of heparin and heparan sulfate with His(111) residue of the cathepsin B occluding loop. These results strongly suggest that heparan sulfate may be an important binding site for cathepsin B at cell surface, reporting a novel physiological role for heparan sulfate proteoglycans.
- Published
- 2001
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22. Hydrolysis by plasma kallikrein of fluorogenic peptides derived from prorenin processing site.
- Author
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Almeida PC, Chagas JR, Cezari MH, Juliano MA, and Juliano L
- Subjects
- Amino Acid Sequence, Animals, Cattle, Humans, Hydrolysis, Molecular Sequence Data, Peptides chemistry, Rats, Sequence Homology, Amino Acid, Enzyme Precursors chemistry, Kallikreins blood, Peptides metabolism, Renin chemistry
- Abstract
Human plasma kallikrein (HPK) activates plasma prorenin to renin, and the physiological significance of this activation is still unknown. In this paper we investigated the efficiency and the cleavage pattern of the hydrolysis by HPK of the internally quenched fluorescent peptides (qf-peptides) derived from the amino acid sequence of human prorenin cleavage site. The peptide Abz-F-S-Q-P-M-K-R-L-T-L-G-N-T-T-Q-EDDnp (Abz=ortho-aminobenzoic acid, and EDDnp=N-[2,4-dinitrophenyl]-ethylene diamine), that corresponds to the amino acid sequence P(7) to P(7)' of human prorenin cleavage site, is hydrolyzed at the correct processing site (R-L bond) with k(cat)/K(m)=85 mM(-1) s(-1). Alanine was scanned in all positions from P(5) to P(5)' in order to investigate the substrate specificity requirements of HPK. The qf-peptides derived from the equivalent segment of rat prorenin, that has Lys-Lys as basic amino acid pair, and the peptide Abz-NVTSPVQ-EDDnp that contains the proposed cleavage site of rat prorenin have very low susceptibility to hydrolysis by rat plasma kallikrein. These data are according to the previously reported absence of rat plasma prorenin activation by rat plasma kallikrein (RPK), and with the view that prorenin activation in rat requires alternative enzymes and/or mechanism. All the obtained peptides described in this paper were also assayed with bovine trypsin that was taken as a reference protease because it is commonly used to activate prorenin.
- Published
- 2000
- Full Text
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23. Characterization of a tissue kallikrein inhibitor isolated from Bauhinia bauhinioides seeds: inhibition of the hydrolysis of kininogen related substrates.
- Author
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Oliva ML, Mendes CR, Juliano MA, Chagas JR, Rosa JC, Greene LJ, Sampaio MU, and Sampaio CA
- Subjects
- Amino Acid Sequence, Hydrolysis drug effects, Molecular Sequence Data, Plant Proteins isolation & purification, Seeds chemistry, Sequence Homology, Amino Acid, Serine Proteinase Inhibitors isolation & purification, Substrate Specificity, Fabaceae chemistry, Kininogens antagonists & inhibitors, Kininogens metabolism, Plant Proteins chemistry, Plants, Medicinal, Serine Proteinase Inhibitors chemistry, Tissue Kallikreins antagonists & inhibitors
- Abstract
Trypsin inhibitors were purified from a saline extract of Bauhinia bauhinioides seeds by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q ion-exchange chromatography or, alternatively, by affinity chromatography on trypsin-Sepharose. Both B. bauhinioides isolated inhibitors, BbTI-I and BbTI-II, inhibit trypsin being the dissociation constant 0.6 and 0.36 nM, respectively. BbTI-II only inhibits porcine pancreatic kallikrein hydrolysis of H-Pro-Phe-Arg-AMC (Ki 2.0 nM); the bradykinin-containing sequence LGMISLMKRPPGFSPFRSSRI-NH2 and the two kininogen related flanking quenched substrates Abz-MISLMKRP-EDDnp (Ki 2.0 nM) and Abz-FRSSRQ-EDDnp (Ki 2.5 nM).
- Published
- 1999
- Full Text
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24. Comparison of human and porcine tissue kallikrein substrate specificities.
- Author
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Del Nery E, Chagas JR, Juliano MA, Juliano L, and Prado ES
- Subjects
- Amino Acid Sequence, Animals, Arginine metabolism, Cattle, Fluorescent Dyes metabolism, Humans, Hydrolysis, Lysine metabolism, Methionine metabolism, Molecular Sequence Data, Oligopeptides metabolism, Peptide Fragments metabolism, Serine metabolism, Species Specificity, Substrate Specificity, Swine, Tissue Kallikreins metabolism
- Abstract
Little is known about the species specificity of tissue kallikrein-kininogen interaction since the kinetic parameters for Lys-bradykinin release from kininogen by tissue kallikreins from different animal species have not been reported. We have now determined the kinetic parameters for hydrolysis by human and porcine tissue kallikrein, hK1 and pK1, respectively (Berg et al., 1992) of two series of intramolecularly quenched fluorogenic peptides having the sequences that flank the scissile Arg-Ser or Met-Lys bond in human and bovine kininogen. Results have shown that peptides having sequences from human kininogen are better substrates for hK1 and peptides derived from bovine kininogen are better substrates for pK1. Kinetic data for hydrolysis of the Arg-Ser bond showed that differences in the interaction of residue(s) in positions P2'-P10' contribute to the efficiency of the cleavage and may be responsible for differences in their susceptibilities to the two kallikreins. Significant variations in the kinetic data were observed for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension at sites P3-P9. The highest k(cat)/Km value in the hydrolysis of Abz-[Gln370-Gln381]-bkng-EDDnp by pk1 demonstrates an important interaction of subsites S5-S4 with Gln and Thr residues in the bovine kininogen segment. A Gln370-Gln391 bovine kininogen fragment used to study the cleavage of both Met-Lys and Arg-Ser bonds in the same molecule confirmed the importance of an extended interaction site for species specificity among tissue kallikreins.
- Published
- 1999
- Full Text
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25. Kininogen-derived fluorogenic substrates for investigating the vasoactive properties of rat tissue kallikreins--identification of a T-kinin-releasing rat kallikrein.
- Author
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El Moujahed A, Brillard-Bourdet M, Juliano MA, Moreau T, Chagas JR, Gutman N, Prado ES, and Gauthier F
- Subjects
- Amino Acid Sequence, Animals, Blood Pressure drug effects, Bradykinin metabolism, Humans, Isoenzymes isolation & purification, Isoenzymes metabolism, Kallikreins isolation & purification, Kallikreins pharmacology, Kinetics, Kininogens metabolism, Male, Molecular Sequence Data, Oligopeptides chemistry, Oligopeptides metabolism, Rats, Rats, Wistar, Sequence Alignment, Submandibular Gland enzymology, Substrate Specificity, Tissue Kallikreins, Bradykinin analogs & derivatives, Kallikreins metabolism, Vasoconstrictor Agents
- Abstract
Peptide substrates with intramolecularly quenched fluorescence that reproduce the rat kininogen sequences at both ends of the bradykinin moiety were synthesized and used to investigate the kinin-releasing properties of five rat tissue kallikreins (rK1, rK2, rK7, rK9, rK10). Substrates derived from rat H- and L-kininogen were cleaved best by rK1, especially that including the N-terminal insertion site of bradykinin, Abz-TSVIRRPQ-EDDnp(Abz = O-aminobenzoyl, EDDnp = ethylenediamine 2,4-dinitrophenyl), which was cleaved at the R-R bond with a k(cat)/Km of 12400 mM(-1) s(-1). Replacement of the P2' residue Pro by Val in Abz-TSVIRRPQ-EDDnp gave a far less specific substrate that was rapidly hydrolysed by all five rat kallikreins and human kallikrein hK1. Peptidyl-N-methyl coumarylamide substrates, which lack prime residues, also had low specificities. The importance of the P2' residue for rK1 specificity was further demonstrated using a human-kininogen-derived substrate that included the N-terminal insertion site of bradykinin (Abz-LMKRP-EDDnp). This was cleaved at the M-K bond by hK1 (kallidin-releasing site), but at the K-R bond (bradykinin-releasing site) by rK1. Competition experiments with Abz-TSVIRRPQ-EDDnp, which is resistant to most kallikreins, and Abz-TSVIRRVQ-EDDnp, a general kallikrein substrate, demonstrated that the former competitively inhibited hydrolysis by rK9 and hK1, with Ki values similar to the Km values for the substrate. Thus Pro in P2' does not prevent the peptide binding to the enzyme active site, but impairs cleavage of the scissile bond. The T-kininogen-derived substrate with the T-kinin C-terminal sequence (Abz-FRLVR-EDDnp) was cleaved by rK10 (k(cat)/Km = 2310 mM(-1) s(-1)) and less rapidly by rK1, rK7 and hK1, at the R-L bond, while that corresponding to the N-terminal (Abz-ALDMMISRP-EDDnp) of T-kinin was resistant to all five kallikreins used, suggesting that none has T-kininogenase activity. But this substrate was hydrolysed by a semipurified sample of submandibular gland extract. Another kallikrein, identified as kallikrein rK3, was isolated from this fraction and shown to hydrolyze Abz-ALDMMISRP-EDDnp; rK3 also specifically released T-kinin from purified T1/T2-kininogen after HPLC fractionation. Injection of purified rK3 and of Abz-ALDMMISRP-EDDnp-cleaving fractions into the circulation of anesthesized rats caused transient falls in blood pressure, as did purified rK1 but none of the other purified rat or human kallikreins. This effect occurred via activation of the kinin system since it was blocked by Hoe140, a kinin receptor antagonist.
- Published
- 1997
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26. Inhibition of cathepsin B by its propeptide: use of overlapping peptides to identify a critical segment.
- Author
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Chagas JR, Ferrer-Di Martino M, Gauthier F, and Lalmanach G
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Cathepsin B chemistry, Cathepsin B metabolism, Enzyme Precursors chemistry, Enzyme Precursors metabolism, Humans, Kinetics, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments metabolism, Rats, Structure-Activity Relationship, Cathepsin B antagonists & inhibitors, Cathepsin B pharmacology, Enzyme Precursors pharmacology, Peptide Fragments pharmacology
- Abstract
Ten overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues 1p-60p) were constructed to identify minimal segments having inhibitory activity towards the mature enzyme, that could be used to develop a new generation of peptide-derived inhibitors specifically targeting the active site of the corresponding proteinase. Three synthetic peptides, containing the pentapeptide Leu-Cys-Gly-Thr-Val (residues 41p-45p) in their sequence, inhibited cathepsin B with Ki values in the micromolar range. Alkylation of the thiol group of Cys-42p of peptide PB8 (36p-50p) resulted in its rapid proteolytic degradation, suggesting that this residue is essential for inhibition. The inhibition constant was slightly improved (Ki = 2 microM) using a longer peptide (26p-50p) which was completely resistant to cleavage even after a prolonged incubation. Alkylation of its cysteinyl residue also resulted in rapid cleavage of the peptide chain. Peptides derived from the rat cathepsin B prosequence also inhibited human cathepsin B with similar Ki values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p-G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PB11 at the Lys-40p-Leu-41p bond, in agreement with the different kinetic properties of these two proteinases. New probes with improved specificity for cysteine proteinases may therefore be designed based on the sequences of their propeptides.
- Published
- 1996
- Full Text
- View/download PDF
27. Met-Lys-bradykinin-Ser, the kinin released from human kininogen by human pepsin.
- Author
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Silva RA, Chagas JR, Juliano L, and Hial V
- Subjects
- Amino Acid Sequence, Animals, Bradykinin chemistry, Bradykinin metabolism, Bradykinin pharmacology, Female, Gastric Mucosa metabolism, Humans, Kininogens chemistry, Mucous Membrane metabolism, Muscle Contraction drug effects, Rats, Uterus drug effects, Uterus physiology, Bradykinin analogs & derivatives, Kininogens drug effects, Kininogens metabolism, Pepsin A pharmacology
- Abstract
This study was carried out to show the site in kininogen, using synthetic substrates, that is cleaved by a purified human pepsin component with a molecular weight of 35,000 daltons. The study used 4 (four) intramolecularly quenched fluorogenic substrates containing N- and C-terminal sequences around the methionyl-lysyl-bradykinin (MLBK) region of kininogen: Abz-LMKRP-Eddnp, Abz-MISLMKRP-Eddnp, Abz-FRSSR-Eddnp and Abz-RPPGFSPFRSSRQ-Eddnp. The hydrolysis on N-terminal sequences Abz-LMKRP-Eddnp and Abz-MISLMKRP-EDDnp occurred at L-M linkage and on C-terminal sequences Abz-FRSSR-Eddnp, and Abz-RPPGFSPFRSSRQ-Eddnp occurred at S-S linkage. The released peptide Abz-RPPGFSPFRS was able to contract rat uterus muscle. The results suggest that Met-Lys-Bradykinin-Ser, should be the peptide released from human kininogen by a purified human pepsin component.
- Published
- 1996
- Full Text
- View/download PDF
28. Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases.
- Author
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Lalmanach G, Serveau C, Brillard-Bourdet M, Chagas JR, Mayer R, Juliano L, and Gauthier F
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Cathepsins antagonists & inhibitors, Cathepsins metabolism, Chromatography, High Pressure Liquid, Consensus Sequence, Cystatins metabolism, Cystatins pharmacology, Cysteine Proteinase Inhibitors pharmacology, Fluorescence, Hydrolysis, Kinetics, Liver enzymology, Molecular Sequence Data, Papain antagonists & inhibitors, Papain metabolism, Peptide Fragments metabolism, Peptide Fragments pharmacology, Rats, Substrate Specificity, Cystatins chemistry, Cysteine Endopeptidases metabolism, Cysteine Proteinase Inhibitors chemical synthesis, Drug Design, Peptide Fragments chemical synthesis
- Abstract
Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzed Abz-QVVAGA-EDDnp and Abz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10(7) M-1 sec-1 range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10(5) M-1 sec-1 range due to lower k(cat) values, with the Km's being identical to those with papain. Only Abz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN2 was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k + 0 = 1,100,000 M-1 sec-1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN2. Thus cystatin-derived peptides provide a valuable frame-work for designing sensitive, selective substrates and inhibitors of cysteine proteinases.
- Published
- 1995
- Full Text
- View/download PDF
29. Substrate specificities of tissue kallikrein and T-kininogenase: their possible role in kininogen processing.
- Author
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Chagas JR, Hirata IY, Juliano MA, Xiong W, Wang C, Chao J, Juliano L, and Prado ES
- Subjects
- Amino Acid Sequence, Animals, Horses, Humans, Hydrolysis, Kinetics, Rats, Substrate Specificity, Swine, Tissue Kallikreins, Kallikreins metabolism, Kininogens metabolism, Protein Processing, Post-Translational
- Abstract
The present studies demonstrate the importance of subsite interactions in determining the cleavage specificities of kallikrein gene family proteinases. The effect of substrate amino acid residues in positions P3-P'3 on the catalytic efficiency of tissue kallikreins (rat, pig, and horse) and T-kininogenase was studied using peptidyl-pNA and intramolecularly quenched fluorogenic peptides as substrates. Kinetic analyses show the different effects of D-amino acid residues at P3, Pro at P'2, and Arg at either P'1 or P'3 on the hydrolysis of substrates by tissue kallikreins from rat and from horse or pig. T-Kininogenase was shown to differ from tissue kallikrein in its interactions at subsites S2, S'1, and S'2. As a result of these differences, Abz-FRSR-EDDnp with Arg at P'2 is a good substrate for tissue kallikreins from horse, pig, and rat but not for T-kininogenase. Abz-FRRP-EDDnp and Abz-FRAPR-EDDnp with Pro at P'2 (rat high molecular weight kininogen sequence) are susceptible to rat tissue kallikrein but not to tissue kallikreins from horse and pig. Arg at P'3 increased the susceptibility of the Arg-Ala bond to rat tissue kallikrein. These data explain the release of bradykinin by rat tissue kallikrein and of kallidin by tissue kallikreins from other animal species. Abz-FRLV-EDDnp and Abz-FRLVR-EDDnp (T-kininogen sequence) are good substrates for T-kininogenase but not for tissue kallikrein. Arg at the leaving group (at either P'1, P'2, or P'3) lowers the Km values of T-kininogenase while Val at P'2 increases its kcat values.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1992
- Full Text
- View/download PDF
30. Intramolecularly quenched fluorogenic peptide substrates for human renin.
- Author
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Oliveira MC, Hirata IY, Chagas JR, Boschcov P, Gomes RA, Figueiredo AF, and Juliano L
- Subjects
- Amino Acid Sequence, Animals, Cathepsin D metabolism, Chromatography, High Pressure Liquid, Fluorometry, Humans, Kinetics, Molecular Sequence Data, Peptides chemical synthesis, Renin antagonists & inhibitors, Renin chemistry, Substrate Specificity, Swine, Peptides metabolism, Renin metabolism
- Abstract
Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.
- Published
- 1992
- Full Text
- View/download PDF
31. Determinants of tissue kallikrein cleavage specificity in the limited proteolysis of kininogens.
- Author
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Prado ES, Chao J, Chagas JR, Juliano MA, and Juliano L
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Horses, Humans, Hydrolysis, In Vitro Techniques, Kinetics, Molecular Sequence Data, Oligopeptides chemistry, Rats, Species Specificity, Substrate Specificity, Swine, Kallikreins metabolism, Kininogens metabolism
- Abstract
Further evidence for interactions at tissue kallikrein extended binding sites, as determinants of the kininogen cleavage specificities is presented. Differences in the cleavage sites in kininogen hydrolysis by rat and other tissue kallikreins is related to subsite S1' specificity, while the low susceptibility of rat kininogen to horse tissue kallikrein is explained by the difference in their subsite S3'.
- Published
- 1992
- Full Text
- View/download PDF
32. Synthesis of human angiotensinogen (1-17) containing one of the putative glycosylation binding sites and its hydrolysis by human renin and porcine pepsin.
- Author
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Hirata IY, Boschcov P, Oliveira MC, Juliano MA, Miranda A, Chagas JR, Tsuboi S, Okada Y, and Juliano L
- Subjects
- Amino Acid Sequence, Angiotensinogen metabolism, Animals, Binding Sites, Chromatography, High Pressure Liquid, Glycosylation, Humans, Kinetics, Molecular Sequence Data, Swine, Angiotensinogen chemistry, Pepsin A metabolism, Renin metabolism
- Abstract
The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH2 ), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH2 with a Km value of 3.4 x 10(-5) M, at pH 7.3 and 37 degrees being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 x 10(-9) mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10-Val11 bond and, surprisingly, His9-Leu10 as well.
- Published
- 1991
- Full Text
- View/download PDF
33. Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins.
- Author
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Chagas JR, Juliano L, and Prado ES
- Subjects
- Animals, Chromatography, High Pressure Liquid, Horses, Hydrolysis, Kallikreins blood, Kallikreins urine, Kinetics, Molecular Sequence Data, Oligopeptides chemical synthesis, Pancreas metabolism, Spectrometry, Fluorescence, Substrate Specificity, Kallikreins metabolism, Oligopeptides metabolism
- Abstract
Five intramolecularly quenched fluorogenic substrates for arginyl hydrolases with the sequence Abz-Phe-Arg-X-Y-EDDnp (X = Arg or Ser; Y = Val, Pro, or Arg) were synthesized by classical solution methods. Kinetics of their hydrolysis by tissue and plasma kallikreins, trypsin, and thrombin characterized Abz-Phe-Arg-Ser-Arg-EDDnp as a specific and sensitive substrate for the continuous assay of tissue kallikreins while Abz-Phe-Arg-Arg-Pro-EDDnp was the best substrate for human plasma kallikrein. The five peptides were poor substrates for trypsin and resistant to thrombin.
- Published
- 1991
- Full Text
- View/download PDF
34. A selective assay for endooligopeptidase A based on the cleavage of fluorogenic substrate structurally related to enkephalin.
- Author
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Juliano L, Chagas JR, Hirata IY, Carmona E, Sucupira M, Oliveira ES, Oliveira EB, and Camargo AC
- Subjects
- Amino Acid Sequence, Animals, Antibodies immunology, Chromatography, High Pressure Liquid, Cysteine Endopeptidases immunology, Hydrolysis, Molecular Sequence Data, Rabbits, Cysteine Endopeptidases analysis, Enkephalins, Ethylenediamines, Fluorescence, Metalloendopeptidases, Oligopeptides
- Abstract
A novel quenched fluorescence substrate, QF-ERP7 (Abz-G-G-F-L-R-R-V-EDDn), structurally related to enkephalins, proved to be suitable for assaying the endooligopeptidase A (E.C.3.4.22.19) activity. The enzyme only splits the L-R bond (Km 1.75 microM, Kcat 8.25 s-1), a reaction efficiently blocked by anti-endooligopeptidase A antibodies and by inhibitor and alternative substrates of the enzyme. Evidences based on the action of inhibitors and on the analysis of QF-ERP7 fragments demonstrated that endooligopeptidase A contributes with 100% of the QF-ERP7 cleaving activity found in the cytosol of rabbit brain homogenates and with 85% of that recovered in the membrane fraction. Homologous substrates, Abz-G-G-F-L-R-R-EDDn and Abz-G-G-F-L-R-EDDn, were resistant to hydrolysis. The convenience and sensitivity of the fluorimetric assay based on the QF-ERP7 moiety offers several advantages compared with previously described painstaking procedures for endooligopeptidase A activity measurements, what will certainly contribute to further our understanding of the role of this enzyme on the peptide hormone metabolism.
- Published
- 1990
- Full Text
- View/download PDF
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