ABSTRACT
A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.
Subject(s)
Plant Proteins/pharmacology , Plasma Kallikrein/antagonists & inhibitors , Rosales/chemistry , Serine Proteinase Inhibitors/isolation & purification , Trypsin Inhibitors/isolation & purification , Amino Acid Sequence , Animals , Binding Sites , Cattle , Chromatography, Affinity , Fluorescent Dyes/metabolism , Humans , Hydrolysis , Kinetics , Molecular Sequence Data , Molecular Weight , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Plant Proteins/isolation & purification , Plasma Kallikrein/metabolism , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/pharmacology , Trypsin/chemistry , Trypsin Inhibitors/genetics , Trypsin Inhibitors/pharmacologyABSTRACT
We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.
Subject(s)
Fluorescent Dyes/pharmacology , Kallikreins/metabolism , Peptides/pharmacology , Plants/chemistry , Trypsin Inhibitor, Kunitz Soybean/pharmacology , Amino Acid Sequence , Animals , Binding Sites/drug effects , Fluorescent Dyes/chemical synthesis , Humans , Hydrolysis , Kallikreins/drug effects , Molecular Sequence Data , Peptides/chemical synthesis , Swine , Trypsin Inhibitor, Kunitz Soybean/isolation & purificationABSTRACT
É apresentado um caso de linfoma linfoblástico da infância, cujo diagnóstico foi suspeitado a partir das alteraçöes radiológicas esqueléticas. O achado de vértebra plana generalizada, pouco mencionado na literatura pertinente, levou os autores à publicaçäo do caso
Subject(s)
Humans , Child , Diagnosis, Differential , Lymphoma/diagnosis , Lumbar Vertebrae/physiopathology , Thoracic Vertebrae/physiopathology , BrazilABSTRACT
1. Mice peritoneal mast cells previously treated with alloantibodies directed against private and public major histocompatibility antigens became partially resistant to subsequent passive sensitization with homologous IgG1-rich serum. 2. Sensitization of the mast cells was evaluated in terms of the percentage of cells that degranulated upon challenge with the specific antigen. 3. The phenomenon was immunologically specific and the responsible alloantigens were shown to be coded by the H-2K or H-2D regions.
Subject(s)
H-2 Antigens/immunology , Histamine Release , Isoantibodies/pharmacology , Mast Cells/immunology , Anaphylaxis/immunology , Animals , Female , Immune Sera , Immunization, Passive , Immunoglobulin G/immunology , Immunologic Capping , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Receptors, ImmunologicABSTRACT
Mice peritoneal mast cells previously treated with alloantibodies directed against private and public major histocompatibility antigens became partially resistant to subsequent passive sensitization with homologous IgG1-rich serum. 2. Sensitization of the mast cells was evaluated in terms of the percentage of cells that de granulated upon challenge with the specifc antigen. 3. The phenomenon was immunologically specific and the responsible alloantigens were shown to be coded by the H-2K or H-2D regions
Subject(s)
Mice , Animals , Female , Immunoglobulin G , Isoantibodies/pharmacology , Mast Cells/drug effects , Mice, Inbred DBA , OvalbuminABSTRACT
The necessity of using A1(OH)3 gel as an adjuvant in the IgE secondary response to a low dose of ovalbumin in mice was studied. Our results demonstrated that a good secondary response can be obtained independent of the presence of A1(OH)3 gel.
Subject(s)
Aluminum Hydroxide/pharmacology , Immunoglobulin E/biosynthesis , Immunologic Memory/drug effects , Adjuvants, Immunologic , Animals , Antigens , Dose-Response Relationship, Immunologic , Female , Mice , Ovalbumin/immunologyABSTRACT
A transient and apparently orderly re-expression of embryonal antigens in planarian regenerates has been demonstrated. This finding seems to corroborate the hypothesis that regeneration in planarians is based on a recapitulation of mechanisms that operate the embryogenesis of these animals, andgives some support to the concept of cancer as a misprogrammed regeneration when contemplated against the background of facts pointed out in the literature.
Subject(s)
Antigens/analysis , Planarians/embryology , Planarians/immunology , Regeneration , Turbellaria/embryology , Turbellaria/immunology , Animals , MiceABSTRACT
Immune responsiveness of inbred mice to low doses of ovalbumin or ovomucoid is under control of single dominant genes closely linked to alleles of the H-2 locus. High responsiveness to ovomucoid is linked with the H-2(a) and H-2(k) alleles, and to ovalbumin with the H-2(b), H-2(d), and H-2(q) alleles.
