RESUMEN
In plant cells, the pyruvate dehydrogenase (PDH) complex that requires lipoic acid as an essential coenzyme is located in plastids and mitochondria. The enzyme complex has to be lipoylated in both organelles. However, the lipoyltransferase located in plastids has not been reported. In this study, an Arabidopsis thaliana LIP2p cDNA for a lipoyltransferase located in plastids has been identified. We have shown that this cDNA encodes a lipoyltransferase by demonstrating its ability to complement an Escherichia coli mutant lacking lipoyltransferase activity, and that LIP2p is targeted into chloroplasts. These findings suggest that LIP2p is located in plastids and responsible for lipoylation of the plastidial PDH complex.
Asunto(s)
Aciltransferasas/genética , Arabidopsis/genética , Plastidios/enzimología , Aciltransferasas/química , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Clonación Molecular , ADN Complementario , Prueba de Complementación Genética , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Lipoic acid is a coenzyme essential to the activity of enzymes such as pyruvate dehydrogenase, which play important roles in central metabolism. However, neither the enzymes responsible for biosynthesis nor the biosynthetic event of lipoic acid has been reported in mammalian cells. In this study, a mouse mLIP1 cDNA for lipoic acid synthase has been identified. We have shown that the cDNA encodes a lipoic acid synthase by its ability to complement a mutant of Escherichia coli defective in lipoic acid synthase and that mLIP1 is targeted into the mitochondria. These findings suggest that mammalian cells are able to synthesize lipoic acid in mitochondria.
Asunto(s)
Mitocondrias/genética , Sulfurtransferasas/genética , Ácido Tióctico/biosíntesis , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Células CHO , Cricetinae , ADN Complementario/aislamiento & purificación , Escherichia coli/genética , Prueba de Complementación Genética , Masculino , Ratones , Ratones Endogámicos ICR , Mitocondrias/enzimología , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Sulfurtransferasas/metabolismoRESUMEN
Lipoic acid is an essential coenzyme required for activity of several key enzyme complexes, such as the pyruvate dehydrogenase complex, in the central metabolism. In these complexes, lipoic acid must be covalently attached to one of the component proteins for it to have biological activity. We report the cloning and characterization of Arabidopsis thaliana LIP2 cDNA for lipoyltransferase that catalyzes the transfer of the lipoyl group from lipoyl-acyl carrier protein to lipoate-dependent enzymes. This cDNA was shown to code for lipoyltransferase by its ability to complement an Escherichia coli lipB null mutant lacking lipoyltransferase activity. The expressed enzyme in the E. coli mutant efficiently complemented the activity of pyruvate dehydrogenase complex, but less efficiently than that of 2-oxoglutarate dehydrogenase complex. Comparison of the deduced amino acid sequence of LIP2 with those of E. coli and yeast lipoyltransferases showed a marked sequence similarity and the presence of a leader sequence presumably required for import into mitochondria. Southern and northern hybridization analyses suggest that LIP2 is a single-copy gene and is expressed as an mRNA of 860 nt in leaves. Western blot analysis with an antibody against lipoyltransferase demonstrated that a 29 kDa form of lipoyltransferase is located in the mitochondrial compartment of A. thaliana.
Asunto(s)
Aciltransferasas/genética , Proteínas Bacterianas , Proteínas de Escherichia coli , Ligasas , Ácido Tióctico/metabolismo , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Secuencia de Bases , Clonación Molecular , ADN de Plantas , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Prueba de Complementación Genética , Líquido Intracelular/metabolismo , Lipoproteínas/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis , Homología de Secuencia de AminoácidoRESUMEN
When type 1 diabetes mellitus was induced in rats by injecting streptozotocin, histidine decarboxylase expression was abnormally up-regulated in a transcriptional level, and 7 d after the injection, the enzyme activity was increased about 3-fold over the control (p < 0.05). When the diabetic rats were administered with insulin for 3 d, the increased histidine decarboxylase activity returned to a normal level in addition to normalization of the plasma glucose level. The plasma gastrin level in the fasting state was also significantly elevated in the diabetic rats, and the insulin treatment normalized the level. In the diabetic rats, fasting gastric acid output increased significantly and gastric pH was lowered. These results suggest that the gastric histidine decarboxylase activity and plasma gastrin level are increased in connection with the depletion of insulin in streptozotocin-induced diabetic rats, and gastric acid secretion is stimulated at a basal level, presumably due to increases in the concentrations of histamine and gastrin in oxyntic mucosa.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Ácido Gástrico/metabolismo , Mucosa Gástrica/enzimología , Gastrinas/sangre , Histidina Descarboxilasa/metabolismo , Animales , Glucemia/metabolismo , Northern Blotting , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Histidina Descarboxilasa/genética , Insulina/administración & dosificación , Intestino Delgado/enzimología , Riñón/enzimología , Hígado/enzimología , Masculino , Páncreas/enzimología , Ratas , Ratas Wistar , Estreptozocina , Regulación hacia ArribaRESUMEN
Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.
Asunto(s)
Arabidopsis/metabolismo , Sulfurtransferasas/genética , Ácido Tióctico/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Escherichia coli/genética , Mutación , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/enzimología , Sulfurtransferasas/química , Sulfurtransferasas/metabolismoRESUMEN
Oxethazaine (OXZ), a potent topical anesthetic, was found to induce red blood cell (RBC) lysis in vitro, depending upon concentrations of OXZ, RBC and Ca2+. In a 2% RBC suspension, 100 microM OXZ caused almost complete hemolysis in the presence of 1.3 mM Ca2+ with only a minimal effect in its absence, while higher concentrations of OXZ (400 microM<) produced hemolysis without Ca2+. The hemolysis induced by OXZ plus Ca2+ was preceded by a rapid increase in 45Ca2+ uptake by RBCs, with both the hemolysis and Ca2+ uptake being inhibited by 1 mM CoCl2, NiCl2, and quinine. Together with the Ca2+ influx, rapid influx of Na+ and efflux of K+ occurred, and an increasing external K+/Na+ concentration ratio inhibited both hemolysis and Ca2+ influx. Morphologically, OXZ plus Ca2+ caused rapid transformation to spheroechinocytes, the formation of blebs and the pinching-off of blebs, whereas OXZ alone produced membrane invagination. SDS-PAGE analysis of the ghosts prepared from the RBCs treated with OXZ plus Ca2+ revealed derangement of cytoskeletal components. OXZ is a rare drug that exhibits a Ca2+ ionophore-like action, increasing the Ca2+ permeability of plasma membranes.