RESUMO
Lysine-oxoglutarate reductase and saccharopine dehydrogenase are enzymic activities that catalyse the first two steps of lysine degradation through the saccharopine pathway in upper eukaryotes. This paper describes the isolation and characterization of a cDNA clone encoding a bifunctional enzyme bearing domains corresponding to these two enzymic activities. We partly purified those activities from mouse liver and showed for the first time that both a bifunctional lysine-oxoglutarate reductase/saccharopine dehydrogenase and a monofunctional saccharopine dehydrogenase are likely to be present in this organ. Northern analyses indicate the existence of two mRNA species in liver and kidney. The longest molecule, 3.4 kb in size, corresponds to the isolated cDNA and encodes the bifunctional enzyme. The 2.4 kb short transcript probably codes for the monofunctional dehydrogenase. Sequence analyses show that the bifunctional enzyme is likely to be a mitochondrial protein. Furthermore, enzymic and expression analyses suggest that lysine-oxoglutarate reductase/saccharopine dehydrogenase levels increase in livers of mice under starvation. Lysine-injected mice also show an increase in lysine-oxoglutarate reductase and saccharopine dehydrogenase levels.
Assuntos
Lisina/análogos & derivados , Lisina/metabolismo , Complexos Multienzimáticos/metabolismo , Sacaropina Desidrogenases/metabolismo , Sequência de Aminoácidos , Animais , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Rim/enzimologia , Fígado/enzimologia , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Sacaropina Desidrogenases/genética , Sacaropina Desidrogenases/isolamento & purificação , Homologia de Sequência de Aminoácidos , Inanição/metabolismo , Distribuição TecidualRESUMO
The lysine-oxoglutarate reductase (LOR) domain of the bifunctional enzyme lysine-oxoglutarate reductase-saccharopine dehydrogenase (LOR/SDH) from maize endosperm was shown to be activated by Ca2+, high salt concentration, organic solvents and Mg2+. The Ca2+-dependent enhancement of LOR activity was inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and calmidazolium. Limited proteolysis was used to assess the structure/function relationship of the enzyme. Digestion with elastase separated the bifunctional 125-kDa polypeptide into two polypeptides of 65 kDa and 57 kDa, containing the functional domains of LOR and SDH, respectively. Proteolysis did not affect SDH activity, while LOR showed a time-dependent and protease-concentration-dependent inactivation followed by reactivation. Prolonged digestion or increasing amounts of elastase produced a complex pattern of limit polypeptides derived from additional cleavage sites within the 65-kDa (LOR) and 57-kDa (SDH) domains. The SDH-containing polypeptides inhibited the enzymatic activity of LOR-containing polypeptides. When separated from the SDH domain by limited proteolysis and ion-exchange chromatography, the LOR domain retained its Ca2+ activation property, but was no longer activated by high salt concentrations. These results suggest that the LOR activity of the native enzyme is normally inhibited such that after modulation, the enzyme undergoes a conformational alteration to expose the catalytic domain for substrate binding.
Assuntos
Sacaropina Desidrogenases/química , Sacaropina Desidrogenases/metabolismo , Zea mays/enzimologia , Cálcio/farmacologia , Cátions Bivalentes/farmacologia , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Cobalto/farmacologia , Ácido Egtázico/farmacologia , Ativação Enzimática , Cinética , Magnésio/farmacologia , Peso Molecular , Concentração Osmolar , Sacaropina Desidrogenases/isolamento & purificação , Sementes/enzimologia , Solventes , Zinco/farmacologiaRESUMO
The seed storage proteins of Coix, sorghum and maize are codified by homologous genes which are coordinately expressed in the endosperm in a temporal-specific fashion. Opaque2 (O2), a bZIP protein originally isolated from maize, has been described as a transcription activator of alpha- and beta-prolamin genes. The isolation and characterization of cDNA and genomic clones encoding the Opaque2 homologue from Coix are reported here. The coding region of the Coix O2 gene is interrupted by five introns and codifies a polypeptide of 408 amino acids. Comparison of the deduced amino acid sequence with two different sequences of maize O2 protein showed that the Coix O2 protein is similar to the maize O2 isolated from W22 maize inbred line. The Coix O2 protein has the same binding specificity and expression pattern of the maize O2. The O2 proteins together with OHP1, OsBZIPPA, SPA, CPRF2 and RITA1 were assigned to one of the five bZIP plant families in an updated classification of plant bZIP according to bZIP domain similarity.
Assuntos
Proteínas de Ligação a DNA/genética , Genes de Plantas , Zíper de Leucina , Poaceae/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Evolução Molecular , Íntrons , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Poaceae/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Zea mays/genéticaRESUMO
The maize opaque 2 (o2) mutation is known to have numerous pleiotropic effects. Some polypeptides have their expression depressed while others are enhanced. The best characterized effects of the o2 mutation are those exerted on endosperm genes encoding the storage protein class of the 22 kDa alpha-zeins and the ribosome inactivating protein b-32. The Opaque 2 (O2) locus encodes a basic domain-leucine zipper DNA-binding factor, O2, which transcriptionally regulates these genes. In the maize-related grass Coix lacryma-jobi, an O2-homologous protein regulates the 25 kDa alpha-coixin family. We show in this paper that O2 transcriptionally regulates the structurally and developmentally different class of the beta-prolamins. A new O2-binding box was identified in beta-prolamin genes from maize and Coix that, together with the boxes previously identified in other endosperm expressed genes, forms a curious collection of O2 cis elements. This may have regulatory implications on the role of O2 in the mechanism that controls coordinated gene expression in the developing endosperm. Considering that the O2 locus controls at least three distinct classes of genes in maize endosperm, we propose that the O2 protein may play a more general role in maize endosperm development than previously conceived.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/biossíntese , Poaceae/genética , Poaceae/metabolismo , Zea mays/genética , Zea mays/metabolismo , Sequência de Bases , Clonagem Molecular , Cotilédone , Proteínas de Ligação a DNA/biossíntese , Genes de Plantas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmídeos , Prolaminas , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
The maize Opaque2 (O2) protein is a "leucine zipper" DNA binding factor that interacts with the sequence TCCACGTAGA in the promoters of the 22-kD alpha-zein genes and activates its transcription. A completely different consensus sequence (GATGAPyPuTGPu) identified in b-32, a gene that encodes an abundant albumin that is also under control of the O2 locus, can also be bound by the O2 protein. We showed that the gene encoding the 22-kD-like alpha-coixin, the alpha-prolamin of the maize-related grass Coix, can also be transactivated by the O2 protein. A binding assay in vitro and footprint analysis demonstrated that the GACATGTC sequence of the alpha-coixin promoter can be recognized and protected by the maize O2 protein. Employing transient expression experiments in immature maize endosperm and tobacco mesophyll protoplasts, we demonstrated that the O2 protein can activate expression of the beta-glucuronidase reporter gene placed under the control of the 22-kD-like alpha-coixin promoter. We also demonstrated that a 22-kD-like alpha-coixin pseudogene promoter is transactivated by the maize O2 protein.