Altered kinetic properties of rat heart mitochondrial enzymes following experimental-thyrotoxicosis

Effects of T_3- and T₄-induced thyrotoxicosis on temperature-dependent Arrhenius kinetics of succinate oxidase and Mg²⁺- and Mg²⁺ + 2,4-dinitrophenol-dependent ATPase activities in rat heart mitochondria were examined, For succinate oxidase system, treatment with T₃ and T₄ caused increase in the energy of activation in high temperature range in a dose-dependent manner. For low temperature range, increase in energy of activation was apparent only with higher doses of the hormones; with low doses a small but reproducible decrease was evinced. The phase transition temperature decreased significantly under these conditions. For the Mg²⁺- and Mg²⁺+2,4-dintro-phenol-dependent-ATPase activities, the activation energy values in high temperature range decreased in general. Activation energy values in low temperature range recorded a generalized increase in the Mg²⁺-ATPase enzyme system while the value did not change significantly for the Mg²⁺ + 2,4-dinitrophenol-ATPase; phase transition temperature registered a small but reproducible decrease under these conditions. The results are suggestive of increased membrane fluidization possibly through increased proportion of unsaturated fatty acids. The differential effects seen for succinate oxidase and ATPase systems are consistent with different lipid protein domains of these enzyme systems.     

Enteric helminth coinfection enhances host susceptibility to neurotropic flaviviruses via a tuft cell-IL-4 receptor signaling axis

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Model-assisted formate dehydrogenase-O (fdoH) gene knockout for enhanced succinate production in Escherichia coli from glucose and glycerol carbon sources

Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H⁺ conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l^?1 (41.2-fold in 7 days) from glycerol and .39 g l^?1 (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.     

Hexose metabolism in pancreatic islets: Succinate dehydrogenase activity in islet homogenates

Succinate dehydrogenase activity was measured in rat pancreatic islet homogenates incubated in the presence of [1,4-¹⁴C]succinate, the reaction velocity being judged through the generation of ¹⁴CO₂ in the auxiliary reactions catalysed by pig heart fumarase and chicken liver NADP-malate dehydrogenase. In the presence of 1·0 mM succinate, the reaction velocity averaged 5·53 ± 0·44 pmol min^?1 μg^?1 islet protein. The K_m for succinate was close to 0·4 mM and the enzymic activity was restricted to mitochondria. These kinetic results indicate that, under the present experimental conditions, the activity of succinate dehydrogenase does not vastly exceed that of either NAD-isocitrate dehydrogenase or the 2-ketoglutarate dehydrogenase complex, at least when the latter enzymes are activated by ADP and/or Ca²⁺. Nevertheless, the activity of succinate dehydrogenase is sufficient to account for the increase in O₂ uptake evoked in intact islets by the monomethyl ester of succinic acid. It could become a rate-limiting step of the Krebs cycle in models of B-cell dysfunction.     

Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Radioactivity eventually destined for the chromatophore membrane of Rhodopseudomonas sphaeroides was shown in pulse-chase studies to appear first in a distinct pigmented fraction. This material formed an upper pigmented band which sedimented more slowly than chromatophores when cell-free extracts were subjected directly to rate-zone sedimentation on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified fraction contained polypeptide bands of the same mobility as light-harvesting bacteriochlorophyll a and reaction center-associated protein components of chromatophores; these were superimposed upon cytoplasmic membrane polypeptides. The pulse-chase relation was confined mainly to the polypeptide components of these pigment-protein complexes. It is suggested that the isolated fraction may be derived from sites at which new membrane invagination is initiated.     

A new cleavable reagent for cross-linking and reversible immobilization of proteins.

We have prepared a new bifunctional reagent for the cross-linking and reversible immobilization of proteins through their amine groups. This compound, ethylene glycolyl bis(succinimidyl succinate), reacts rapidly with proteins, at pH 7 and at high dilution. The resulting protein cross-links are readily cleaved at pH 8.5 using hydroxylamine for 3–6 hr. at 37°C. Substantial enzymatic activity was observed with lactic dehydrogenase after such reversible cross-linking. Trypsin immobilized on agarose using this reagent retains full specific activity, is stable for weeks in the cold, and may be released with hydroxylamine at 25°C. This compound appears suitable for studies involving proteins with essential disulfide linkages.     

Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells

Alpha-tocopheryl succinate (alpha-TOS), a redox-silent analogue of vitamin E, induces apoptosis in multiple cell lines in a selective manner, by activating the intrinsic pathway. Since it is a highly hydrophobic compound, it may require a carrier protein for its trafficking to intracellular targets like mitochondria. We studied the role of the ubiquitous tocopherol-associated protein-1 (TAP1 or sec14-like 2) in apoptosis induction by alpha-TOS in malignant mesothelioma (MM) cells. Over-expression of TAP1 in MM cells sensitised them to apoptosis by low doses of alpha-TOS which were sub-apoptotic for the parental cells. Apoptosis induced in TAP1-over-expressing cells was mitochondria- and caspase-dependent, as suggested by dissipation of mitochondrial trans-membrane potential and inhibition by zVAD-fmk, respectively. Binding assays showed affinity of alpha-TOS for TAP1. Finally, TAP1 over-expressing cells accumulated alpha-TOS at higher levels compared to their normal counterparts. We suggest that TAP1 may act as an intracellular shuttle for alpha-TOS, promoting apoptosis initiated by this vitamin E analogue, as shown here for MM cells.