RESUMO
The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.
Assuntos
Proteínas de Bactérias/metabolismo , Colletotrichum/enzimologia , Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Albuminas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Endopeptidases/química , Endopeptidases/isolamento & purificação , Espaço Extracelular/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Globulinas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Especificidade por Substrato , TemperaturaRESUMO
It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.
