Characteristics of 2-oxoglutarate and glutamate transport in spinach chloroplasts

The transport of glutamate, 2-oxoglutarate and malate in intact spinach chloroplasts was determined using a double-silicone-layer centrifugation technique in which the silicone layers stayed separated at the end of centrifugation. Glutamate was found to be transported via the dicarboxylate but not the 2-oxoglutarate translocator. Hence the kinetic parameters (i.e.K _m,K _i andV _max) determined in glutamate-preloaded chloroplasts represent the kinetic constants of the dicarboxylate translocator. Measurements from malate- or succinate-preloaded chloroplasts represent the aggregate values of both the dicarboxylate and the 2-oxoglutarate translocators. Calculations showed that the 2-oxoglutarate and glutamate transport required to support the high fluxes of photorespiratory NH₃ recycling could be achieved if the transport of these two dicarboxylates occurred on separate translocators. It is proposed that during photorespiration the transport of 2-oxoglutarate into and glutamate out of the chloroplast occurred via the 2-oxoglutarate and the dicarboxylate translocators, respectively. These transports are coupled to malate counter-exchange in a cascade-like manner resulting in a net 2-oxoglutarate/glutamate exchange with no net malate uptake.Abbreviation 2-OG 2-oxoglutarate     

Protein translocationin vitro: Biochemical characterization of genetically defined translocation components

Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation inE. coli. The powerful combination of these approaches is exemplified in the use of anin vitro protein synthesis-protein translocaltion system to analyze the role of genetically defined components of the protein translocation machinery. We describe in this review recent results focusing on the function of thesecA, secB, andsecY gene products and the demonstration of their requirement forin vitro protein translocation. The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase. We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or protein-ATPase.     

The Cyanelles (Organelles of a Low Evolutionary Scale) Possess a Phosphate-Translocator and a Glucose-Carrier in Cyanophora paradoxa

Cyanelles from Cyanophora paradoxa can easily be isolated and assayed for their carrier composition by the silicone oil filtering technique. The present investigation demonstrates a P_i-translocator transferring phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate in a counter exchange mode in cyanelles as in chloroplasts of higher plants. The uptake of P_i is inhibited by dihydroxyacetone phosphate, phosphoglycerate and glucose-6-P, only poorly by phosphoenolpyruvate and not by 2-phosphoglycerate. The inhibitors pyridoxalphosphate and 4,4′diisothiocyanostilbene-2,2K'disulfonic acid at low concentration also affect P_i-uptake. Cyanelles probably transport photosynthate (reductant and ATP) by triosephosphates. This is the first demonstration of a phosphate translocator in an organism of a low evolutionary scale. Cyanelles also transport glucose which proceeds in two phases. In the lower concentration range (≤ 2.5 mM), glucose penetrates by facilitated diffusion, whereas transport follows first-order kinetics at higher amounts (> 2.5 mM). In the low concentration range, glucose-transport is affected by high concentrations of 3-O-methylglucose and fructose. The physiological role of the glucose-transport carrier in Cyanophora is doubtful. It may function in transporting glucose into cyanelles if the carbon level inside them becomes limiting, e.g. in dark periods.     

Physiological factors determining formation of plastocyanin and plastidic cytochrome c-553 in Scenedesmus

Physiological conditions necessary for the formation of plastocyanin and the concurrent cessation of cytochrome c-553 formation were studied in cells of copper-deficient Scenedesmus acutus after the addition of copper. Plastocyanin is formed after a lag-phase, leaving constant the content of plastidic cytochrome c-553. Therefore, the concentration of plastocyanin per cell increases and the concentration of cytochrome c-553 decreases during growth. Formation of plastocyanin during the induction period studied is dependent on light intensity. In the dark, there is a 90% inhibition, whereas under light intensities above 50 Wm^-2, a ratio of 1.3 molecules plastocyanin per 1,000 molecules chlorophyll is attained.Plastocyanin formations is inhibited by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), but not by moderate concentrations of 3-[3,4-dichlorophenyl]-1,1-dimethylurea (DCMU), and by keeping the algae under a nitrogen atmosphere without CO₂. Concurrently, the cultures treated with FCCP show a decreased endogenous ATP level. The ATP is necessary for plastocyanin formation.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DCMU 3-[3,4-dichlorophenyl]-1,1-dimethylurea - pcv packed cell volume     

2-O-β-d-Glucopyranosyl-carboxyatractyligenin from Coffea L. inhibits adenine nucleotide translocase in isolated mitochondria but is quantitatively degraded during coffee roasting

Atractyloside (1) and carboxyatractyloside (2) are well-known inhibitors of the adenine nucleotide translocase (ANT) in mitochondria, thus effectively blocking oxidative phosphorylation. Structurally related derivatives atractyligenin (3), 2-O-β-d-glucopyranosyl-atractyligenin (4), 3′-O-β-d-glucopyranosyl-2′-O-isovaleryl-2β-(2-desoxy-atractyligenin)-β-d-glucopyranoside (5), and 2-O-β-d-glucopyranosyl-carboxyatractyligenin (6) were isolated from raw beans of Coffea L. and the impact of 1–6 on ANT activity was evaluated in isolated mitochondria. Among the coffee components, 6 significantly inhibited ANT activity leading to reduced respiration. Quantitative analysis in commercial coffees, experimental roastings of coffee, and model experiments using purified compound 6 consistently revealed a complete degradation during thermal treatment. In comparison, raw coffee extracts were found to contain high levels of 6, which are therefore expected to be present in food products enriched with raw coffee extracts. This implies the necessity of analytically controlling the levels of 6 in raw coffee extracts when used as additives for food products.     

The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier

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Nr5a1-Cre-mediated Tspo conditional knockout mice with low growth rate and prediabetes symptoms – A mouse model of stress diabetes

Translocator protein (TSPO) is a high-affinity cholesterol- and drug-binding mitochondrial protein. Nuclear receptor subfamily 5 group A member 1 or steroidogenic factor 1 (Nr5a1)-Cre mice were previously used to generate steroidogenic cell-specific Tspo gene conditional knockout (cKO) mice. TSPO-depleted homozygotes showed no response to adrenocorticotropic hormone (ACTH) in stimulating adrenal cortex corticosterone production but showed increased epinephrine synthesis in the medulla. No other phenotype was observed under normal growth conditions. During these studies, we noted that pairing two cKO mice resulted in the generation of small pups. These pups showed low growth rate at weaning, which has been linked to the development of type 2 diabetes (T2D) in adulthood. Experimental verification of T2D symptoms via blood testing of the adult mice, including glycated hemoglobin and insulin C-peptide measurements, showed that these Tspo cKO mice exhibited sustained hyperglycemia, a sign of prediabetes, likely due to the augmentation of hepatic glucose production mediated by the increased epinephrine. We also observed increased expression of the S100a8 gene, which is upregulated after chronic glucose stimulation. Taken together, the observed prediabetes phenotype and lack of response to ACTH indicate that Tspo cKO mice (Nr5a1-Cre^+/?, Tspo^fl/fl) could provide a useful model to study the link between diabetes and stress.     

Streptomyces genes involved in aerial mycelium formation

Abstract Cloning of genes as suppressors of the aerial mycelium-defective phenotype of Streptomyces griseus HH1 resulting from A-factor-deficiency has led to the identification of several genes, including amfR, amfAlamfB, amfC , and orf1590 . These genes are involved in aerial mycelium formation independent of secondary metabolic function. Among these, AmfR which belongs to the family of response regulators of two-component regulatory systems and AmfA/AmfB similar to ATP-dependent membrane translocators are analogous to the multicomponent phosphorelay and the Spo0K system, respectively, both of which are required for the initiation of sporulation in Bacillus subtilis . Involvement of a protein serine/threonine kinase in aerial mycelium formation is also suggested, because the Streptomyces coelicolor A3(2) afsK gene encoding a 'eukaryotic'-type protein kinase reverses the aerial mycelium-defective phenotype of strain HH1, independent of secondary metabolic function.     

Oxygen consumption and expression of the adenine nucleotide translocator in cells lacking mitochondrial DNA

It has been shown previously that human rho degrees cells, deprived of mitochondrial DNA and consequently of functional oxidative phosphorylation, maintain a mitochondrial membrane potential, which is necessary for their growth. The goal of our study was to determine the precise origin of this membrane potential in three rho degrees cell lines originating from the human HepG2, 143B, and HeLa S3 cell lines. Residual cyanide-sensitive oxygen consumption suggests the persistence of residual mitochondrial respiratory chain activity, about 8% of that of the corresponding parental cells. The fluorescence emitted by the three rho degrees cell lines in the presence of a mitochondrial specific fluorochrome was partially reduced by a protonophore, suggesting the existence of a proton gradient. The mitochondrial membrane potential is maintained both by a residual proton gradient (up to 45 to 50% of the potential) and by other ion movements such as the glycolytic ATP(4-) to mitochondrial ADP(3-) exchange. The ANT2 gene, encoding isoform 2 of the adenine nucleotide translocator, is overexpressed in rho degrees HepG2 and 143B cells strongly dependent on glycolytic ATP synthesis, as compared to the corresponding parental cells, which present a more oxidative metabolism. In rho degrees HeLa S3 cells, originating from the HeLa S3 cell line, which already displays a glycolytic energy status, ANT2 gene expression was not higher as in parental cells. Mitochondrial oxygen consumption and ANT2 gene overexpression vary in opposite ways and this suggests that these two parameters have complementary roles in the maintenance of the mitochondrial membrane potential in rho degrees cells.