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M S Araujo

Publications and source records attributed to M S Araujo.

12 recordsLinked to original sources
PubMed2004

Purification and preliminary characterization of a plasma kallikrein inhibitor isolated from sea hares Aplysia dactylomela Rang, 1828.

An inhibitor active against pancreatic trypsin was found in the crude extract from the sea hares Aplysia dactylomelaRang, 1828. A stronger inhibitory activity against human plasma kallikrein was detectable after treating this extract at 60 degrees C, for 30 min. The plasma kallikrein inhibitor (AdKI) purification was achieved by acetone fractionation (80%) v/v, ion-exchange chromatography on Mono Q column and gel filtration chromatography on Superdex 75 column (FPLC system). By the latter a molecular mass of 2900 Da was estimated. The purified inhibitor strongly inhibits human plasma kallikrein with a K(i) value of 2.2 x 10(-10)M, while human plasmin and pancreatic trypsin were inhibited with K(i) values of 1.8 x 10(-9) and 4.7 x 10(-9)M, respectively. Chymotrypsin, pancreatic elastase, pancreatic kallikrein and thrombin are not inhibited. The effect of AdKI on plasma kallikrein was confirmed by the prolongation of activated partial thromboplastin time, using a clotting time assay. The inhibitor did not affect prothrombin time or thrombin time. AdKi is a more specific inhibitor than other serine proteinase inhibitors from marine invertebrates.

Animals↗
PubMed2003

Mapping of human plasma kallikrein active site by design of peptides based on modifications of a Kazal-type inhibitor reactive site.

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.

Amino Acid Sequence↗
PubMed2000

Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema.

A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.

Amino Acid Sequence↗
PubMed1999

Preliminary characterization of a Kazal-type serine protease inhibitor from Caiman crocodilus yacare plasma.

Blood serine protease inhibitors are becoming better understood and increasingly applied in blood clotting, cancer and other diseases. Reptiles are suitable models for blood coagulation and related processes, moreover, caiman is a good comparative model of a non-poisonous reptile. Recently, we reported the purification of a kininogen, the presence of proteases involved in blood clotting, and a serine protease inhibitor in Caiman crocodilus yacare plasma. In this paper, we described the partial sequence of an inhibitor (CcTI). The inhibitor is an 80-kDa protein, and it inactivates trypsin and chymotrypsin the hydrolysis of specific chromogenic substrates and in the degradation of gelatin. The inhibitor is member of Kazal-type inhibitor family and consists of several domains, its putative reactive site is Arg-His.

Alligators and Crocodiles↗
PubMed1996

Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds.

A trypsin inhibitor was isolated from Enterolobium contortisiliquum seeds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). The amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxymethylated protein and also of purified peptides derived from the protein by cleavage with iodosobenzoic acid and by enzymic digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI contains 174 amino acid residues in two polypeptide chains, an alpha-chain consisting of 134 residues and a beta-chain made up of 40 residues. The inhibitor displays a high degree of sequence identity with other Kunitz-type proteinase inhibitors isolated from the Mimosoideae subfamily. The reactive site was identified (by homology) as the arginine-isoleucine peptide bond at position 64-65. ECTI inhibits trypsin and chymotrypsin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma kallikrein and plasmin, but not thrombin and Factor Xa.

Amino Acid Sequence↗
PubMed1994

Isolation and partial characterization of an endopeptidase from Enterolobium contortisiliquum seeds.

1. Aqueous extracts of Enterolobium contortisiliquum seeds contain an endopeptidase of M(r) 60,000 with specificity for basic amino acid residues. The enzyme was purified by chromatography on DEAE Sephadex, followed by gel filtration on Sephadex G-75 and affinity chromatography on Zinc-Sepharose. The overall purification was 300-fold and the yield about 46%. 2. The endopeptidase hydrolyzes benzoyl-arginine-p-nitroanilide (Bz-Arg-pNan) and acetyl-phenylalanine-arginine-p-nitroanilide (Ac-Phe-Arg-pNan) with Km 14.4 mM and 0.062 mM, respectively. Succinyl-phenylalanine-p-nitroanilide (Suc-Phe-pNan) and tosyl-arginine methyl ester (TAME) were not hydrolyzed. E. contortisiliquum endopeptidase also cleaves a seed protein of low molecular weight from the same E. contortisiliquum seeds, and converts Met-Lys-bradykinin into bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). 3. Metals (1.0 mM) such as Cr3+, Fe3+ and Zn2+ ions inactivate the enzyme when Bz-Arg-pNan was the substrate. Enzyme activity is abolished by EDTA but is partially restored by Cu2+, Al3+, Ba2+, Mn2+, Mg2+, Fe2+, Ca2+ and Co2+ ions. The endopeptidase is not inhibited by the previously purified E. contortisiliquum inhibitors of trypsin and cysteine proteinases, or by soybean trypsin inhibitor (Oliva et al. (1987). Brazilian Journal of Medical and Biological Research, 20:767-770).

Amino Acid Sequence↗
PubMed1992

Caiman kininogen-like cysteine proteinase inhibitor.

Kininogens are the major mammalian plasma cysteine proteinase inhibitors; a kininogen-like protein was also found in the snake Bothrops jararaca plasma. This communication describes a kininogen-like protein in plasma of Caiman crocodilus vacare. Caiman crude plasma, unlike snake plasma, contains a detectable cysteine proteinase inhibitor. The inhibitor was purified by DEAE-Sephadex ion-exchange chromatography and chromatography on carboxy-methylated-papain-Sepharose. The estimated molecular weight of Caiman cysteine proteinase inhibitor is 70,000. Caiman plasma also hydrolyzes plasma kallikrein synthetic substrates and inhibits trypsin. Reptilian kininogen may lack the site for interaction with plasma prokallikrein, and the sequence of the released kinin may be distinct from bradykinin. The poor effectiveness of bradykinin on reptile smooth muscle shows that the reptile kinin receptors may be adapted to a specific kinin.

Animals↗