RESUMO
The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S524), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S524, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S524, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S524 and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.
Assuntos
Encéfalo/enzimologia , Liases Intramoleculares/metabolismo , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Animais , Anticorpos/química , Intestinos/enzimologia , Liases Intramoleculares/química , Liases Intramoleculares/imunologia , Isoenzimas/metabolismo , Masculino , Peso Molecular , Especificidade de Órgãos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fosfoproteínas/química , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Serina/química , Serina/metabolismo , Testículo/enzimologiaRESUMO
Polyclonal antibodies were developed against the flavonoid biosynthetic enzymes, CHS, CHI, F3H, FLS, and LDOX from Arabidopsis thaliana. These antibodies were used to perform the first detailed analysis of coordinate expression of flavonoid metabolism at the protein level. The pattern of flavonoid enzyme expression over the course of seedling development was consistent with previous studies indicating that chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS) are encoded by 'early' genes while leucoanthocyanidin dioxygenase (LDOX) is encoded by a 'late' gene. This sequential expression may underlie the variations in flavonoid end-products produced during this developmental stage, as determined by HPLC analysis, which includes a shift in the ratio of the flavonols, quercetin and kaempferol. Moreover, immunoblot and HPLC analyses revealed that several transparent testa lines blocked at intermediate steps of the flavonoid pathway actually accumulated higher levels of specific flavonoid enzymes and end-products. These results suggest that specific intermediates may act as inducers of flavonoid metabolism.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Flavonoides/biossíntese , Genes de Plantas , Quempferóis , Proteínas de Plantas , Aciltransferases/genética , Aciltransferases/imunologia , Aciltransferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Sequência de Bases , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Glicosídeos/isolamento & purificação , Liases Intramoleculares/genética , Liases Intramoleculares/imunologia , Liases Intramoleculares/metabolismo , Luz , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/imunologia , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Mutação , Oxirredutases/genética , Oxirredutases/imunologia , Oxirredutases/metabolismo , Oxigenases/genética , Oxigenases/imunologia , Oxigenases/metabolismo , Quercetina/análogos & derivados , Quercetina/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.
