RESUMO
We have previously reported that phosphorylation of a 15-kDa protein increased after blue-light irradiation in Neurospora crassa. In this study, the 15-kDa protein was purified using four columns; DEAE-cellulose, Blue-Sepharose, SP-Sepharose and Mono Q. The 15-kDa protein was shown to be homologous with nucleoside diphosphate kinase by amino acid sequencing and was also shown to possess nucleoside diphosphate kinase activity. A gene encoding N. crassa nucleoside diphosphate kinase, ndk-1, was isolated from the mycelial cDNA and genomic libraries. The deduced amino acid sequence of NDK-1 was identical to that of the 15-kDa protein. Northern blot analysis suggested that WC-1 and WC-2, the key factors of blue-light signal transduction in N. crassa, did not regulate NDK-1 at the transcriptional level. NDK-1 also showed rapid autophosphorylation activity and protein kinase activity against myelin basic protein with a Km value of 0.36 mM. These results suggest that NDK-1 acts as a signal transducer by phosphorylating proteins.
Assuntos
Neurospora crassa/enzimologia , Núcleosídeo-Difosfato Quinase/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Humanos , Cinética , Luz , Dados de Sequência Molecular , Peso Molecular , Neurospora crassa/genética , Neurospora crassa/efeitos da radiação , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Fosforilação , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
A simple but accurate method for measuring glucuronoxylan xylanohydrolase activity was developed using coleoptile cell wall particles prepared from maize (Zea mays L.). Two isozymes of glucuronoxylan xylanohydrolases designated as GX1 and GX2 (EC 3.2.1.136) were purified from a commercially available amylase preparation to electrophoretic homogeneity by three cation exchange chromatography steps. Upon characterization no significant differences between the two enzymes were detected: the molecular mass measured by MALDITOF mass spectrometry was 44,360 +/- 100 for GX1 and 44,370 +/- 50 for GX2 suggesting no difference in the total number of amino acid residues. Furthermore the N-terminal amino acid sequence for each of the isozymes was identical through the 37th amino acid residue. The values of pI were determined to be 9.0 for GX1 and 9.1 for GX2. The sensitivity to temperature, pH and to ionic strength was similar for both isozymes as were kinetic parameters including Km and Vmax. No differences could be detected in substrate specificity.