ABSTRACT
Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.
Subject(s)
Adenosine Triphosphate/metabolism , Azospirillum brasilense/enzymology , Bacterial Proteins/metabolism , Ketoglutaric Acids/metabolism , Transcription Factors/metabolism , beta-Galactosidase/metabolism , Azospirillum brasilense/metabolism , Genetic Vectors , PlasmidsABSTRACT
Detached chickpea inflorescences bearing pods at 20 days after flowering (DAF) were cultured for 5 days in complete liquid medium supplemented separately with asparate, myo-inositol, alpha-ketoglutarate and phytic acid. Effect of these metabolites on sugar interconvestion and starch and protein accumulation in developing pods was studied. Substituting asparate (62.5 mM) for glutamine in culture medium decreased relative proportion of sucrose in all pod tissues but increased the level of sugars, starch and protein in pod wall and cotyledons. In cotyledons, whereas myo-inositol (75 mM) reduced the accumulation of starch without affecting protein level, alpha-ketoglutarate (44 mM) increased both starch and protein accumulation. Both myo-inositol and alpha-ketoglutarate increased relative proportion of sucrose in cotyledons. Phytic acid (1 mM) decreased in cotyledons 14C incorporation from glucose into EtOH extract (principally constituted by sugars), amino acids and proteins but increased the same into starch. In cotyledons, phytic acid also increased 14C incorporation from glutamate into amino acids but this increase was negatively correlated with protein synthesis. Phytic acid decreased the relative distribution of 14C from glucose and glutamate into sucrose from pod wall but enhanced the same into EtOH extract from embryo. Based on the results, it is suggested that mode of metabolic response to exogenously supplied metabolites widely differs in pod tissues of chickpea.
Subject(s)
Aspartic Acid/metabolism , Carbohydrate Metabolism , Culture Media/pharmacology , Fabaceae/metabolism , Humans , Inositol/metabolism , Ketoglutaric Acids/metabolism , Phytic Acid/metabolism , Plant Proteins/metabolism , Plants, Medicinal , Seeds/metabolism , Starch/metabolismABSTRACT
The stimulation of the oxyntic cell by gastric secretagogues is associated to the activation of oxidative metabolism. The mechanisms of this activation are not exactly known. In the present work, we investigated the possible direct effect of Ca2+ on both oxoglutarate oxidation in rabbit gastric glands and oxoglutarate dehydrogenase activity (OGDH) is isolated gastric mitochondria. Both carbachol and Ca2+ ionophores +(A23187 and ionomycin) significantly stimulated oxoglutarate oxidation in a dose-dependent manner. This effect was only observed in the presence of low substrate concentrations. BAPTA-AM, an intracellular calcium chelator, significantly inhibited the rate of oxoglutarate oxidation. OGDH activity was stimulated by physiological concentrations of Ca2+ in a dose-depentent manner. This effect was reduced by ruthenium red, an inhibitor of mitochondrial Ca2+ uptake, and it was additionally increased by spermine, a stimulant of Ca2+ influx to the mitochondria. Results support the hypothesis that Ca2+ plays a direct role in the activation of oxidative metabolism in the oxyntic cell
Subject(s)
Animals , Rabbits , Ketoglutaric Acids/metabolism , Calcium/physiology , Gastric Mucosa/metabolism , Oxidoreductases/metabolism , Carbachol/pharmacology , Parietal Cells, Gastric , Parietal Cells, Gastric/metabolism , Ionophores/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Gastric Mucosa , Gastric Mucosa/enzymologyABSTRACT
The stimulation of oxyntic cell by gastric secretagogues is associated to the activation of oxidative metabolism. The mechanisms of this activation are not exactly known. In the present work, we investigated the possible direct effect of Ca2+ on both oxoglutarate oxidation in rabbit gastric glands and oxoglutarate dehydrogenase activity (OGDH) in isolated gastric mitochondria. Both carbachol and Ca2+ ionophores (A23187 and ionomycin) significantly stimulated oxoglutarate oxidation in a dose-dependent manner. This effect was only observed in the presence of low substrate concentrations. APTA-AM, an intracellular calcium chelator, significantly inhibited the rate of oxoglutarate oxidation. OGDH activity was stimulated by physiological concentrations of Ca2+ in a dose-dependent manner. This effect was reduced by ruthenium red, an inhibitor of mitochondrial Ca2+ uptake, and it was additionally increased by spermine, a stimulant of Ca2+ influx to the mitochondria. The results support the hypothesis that Ca2+ plays a direct role in the activation of oxidative metabolism in the oxyntic cell