RESUMO
The epithiospecifier protein (ESP) is a myrosinase (MYR) cofactor, which is necessary to drive the MYR-catalyzed hydrolysis of some specific glucosinolates towards the production of cyanoepithioalkanes instead of isothiocyanates and nitriles. ESP was isolated from Brassica napus seeds by anionic exchange and gel filtration chromatography. ESP showed a molecular weight of about 39 kDa and pI 5.3. The amino acid sequence of several tryptic peptides of ESP (accounting for about 50% of the total sequence) made it possible to establish the high similarity (81% identity) with a hypothetical 37 kDa protein (TrEMBL data base accession number Q39104) and several jasmonate-inducible proteins from Arabidopsis thaliana. This observation suggests that ESP is likely to be involved in jasmonate-mediated defence and disease resistance mechanisms.
Assuntos
Brassica , Óleos de Plantas/química , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Cromatografia em Gel , Cromatografia por Troca Iônica , Ácidos Graxos Monoinsaturados , Glicosídeo Hidrolases/química , Dados de Sequência Molecular , Proteínas de Plantas/química , Óleo de Brassica napus , Alinhamento de SequênciaRESUMO
A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed (Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin with apparent dissociation constants of 3.0 x 10(-10) M and 4.1 x 10(-7) M, at pH 8.0 and 21 degrees C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1:1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M(r) of about 6.7 kDa. The P1-P1' reactive site bond has been tentatively identified at position Arg20-Ile21. RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors.
Assuntos
Brassica/química , Proteínas de Plantas/isolamento & purificação , Inibidores da Tripsina/isolamento & purificação , Sequência de Aminoácidos , Sítios de Ligação/genética , Brassica/genética , Dados de Sequência Molecular , Mostardeira/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Medicinais , Sementes/química , Homologia de Sequência de Aminoácidos , Inibidores da Tripsina/química , Inibidores da Tripsina/genéticaRESUMO
A new serine proteinase inhibitor, mustard trypsin inhibitor 2 (MTI-2), has been isolated from white mustard (Sinapis alba L.) seed by affinity chromatography and reverse phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin, with dissociation constants (Kd) of 1.6 x 10(-10) M and 5.0 x 10(-7) M, respectively, at pH 8.0 and 21 degrees C, the stoichiometry of both proteinase-inhibitor complexes being 1:1. The amino acid sequence of MTI-2, which was determined following S-pyridylethylation, is comprised of 63 residues, corresponding to a molecular weight of about 7 kDa, and shows only extremely limited homology to other serine proteinase inhibitors.
Assuntos
Mostardeira/química , Plantas Medicinais , Sementes/química , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Quimotripsina/efeitos dos fármacos , Relação Dose-Resposta a Droga , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Homologia de Sequência de Aminoácidos , Tripsina/efeitos dos fármacosRESUMO
A major allergen, the Parj I, was purified to homogeneity from Parietaria judaica pollen by means of ultrafiltration dialysis, preparative polyacrylamide gel chromatography and affinity chromatography through a column of Sepharose-monoclonal antibody specific for Parj I. The homogeneity of the Parj I was assessed by one single arc of immunoprecipitation both in cross immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis, by one single band of radiostaining after a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose and by one single peak after a size exclusion chromatography on high-performance liquid chromatography (HPLC). The homogeneity was further supported by crossed Laurell immunoelectrophoretic analysis, in that only one arc of precipitation was magnified in CIE after addition of the purified allergen. The purified Parj I allergen was capable of interacting in vitro with 70% of the human IgE specific for a crude P. judaica extract, as determined by radioallergosorbent test inhibition. The purified Parj I was capable of inducing positive reactions in vivo in skin prick tests, and of inducing release of histamine from blood containing basophils as determined by a histamine release assay. The amino acid analysis of the Parj I showed 118 amino acid residues per monomer analyzed and, among other residues, three methionine residues were detected. The molecular weight of the Parj I estimated by HPLC and amino acid composition was 26 kilodaltons.