http://www.biochemj.org Biochemical Journal RSS feed -- BJ Energy Immediate Publications 0264-6021 1470-8728 Biochemical Journal http://www.biochemj.org/images/BJ_Name.gif http://www.biochemj.org http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120118 I Scott, B R. Webster, J H. Li, M N. Sack 2012-02-06T15:07:51Z doi:10.1042/BJ20120118 Portland Press Limited 2012-02-06 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110765 2+ (Ca²⁺_cyt) (S_0.5 = 225 ± 22 nM) through its effects on aralar. We now also demonstrate that the a-glycerophosphate-dependent respiration is controlled by Ca²⁺_cyt (S_0.5 = 60 ± 10 nM). At higher Ca²⁺_cyt (< 600 nM), BM accumulate Ca²⁺ which enhances the rate of action of intramitochondrial dehydrogenases. The highest Ca²⁺-induced increments of state 3 respiration decrease with substrate in the order glutamate < a-ketoglutarate < isocitrate < a-glycerophosphate < pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca²⁺_cyt, we show that the formation of pyruvate is tightly controlled by Ca²⁺_cyt. Through its common substrate couple NADH/NAD⁺, the formation of pyruvate by lactate dehydrogenase (LDH) is linked to the malate–aspartate shuttle (MAS) with aralar as a central component. A rise of Ca²⁺_cyt in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to five-fold enhancement of OXPHOS rates that is due to an increased substrate supply, acting in a manner similar to a “gas pedal”. In contrast, mitochondrial Ca²⁺ (Ca²⁺_mit) regulates the oxidation rates of substrates which are present within mitochondrial matrix. We postulate that Ca²⁺_cyt is a key factor in adjusting the mitochondrial energisation to the requirements of intact neurons.]]> F N Gellerich, Z Gizatullina, S Trumbeckaite, B Korzeniewski, T Gaynutdinov, E Seppet, S Vielhaber, H Heinze, F Striggow 2012-02-01T16:25:47Z doi:10.1042/BJ20110765 Portland Press Limited 2012-02-01 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111728 S Matsumoto, T Uchiumi, H Tanamachi, T Saito, M Yagi, S Takazaki, T Kanki, D Kang 2012-01-27T11:58:49Z doi:10.1042/BJ20111728 Portland Press Limited 2012-01-27 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111889 2-producing enzyme and a lipid-transporter. L-PGDS is a member of the lipocalin superfamily and can bind a wide variety of lipophilic molecules. Here we show the protective effect of L-PGDS on H₂O₂-induced apoptosis in neuroblastoma cell line SH-SY5Y. L-PGDS expression was increased in H₂O₂-treated neuronal cells, and the L-PGDS level was highly associated with H₂O₂-induced apoptosis, indicating that L-PGDS protected the neuronal cells against H₂O₂-mediated cell death. Cell viability assay revealed that L-PGDS protected against H₂O₂-induced cell death in a concentration-dependent manner. Further, the titration of free-thiols in H₂O₂-treated L-PGDS revealed that H₂O₂ reacted with the thiol of Cys65 of L-PGDS. The MALDI-TOF MS spectrum of H₂O₂-treated L-PGDS showed a 32-Da increase in the mass relative to that of the untreated protein, showing that the thiol was oxidized to sulfinic acid. The binding affinities of oxidized L-PGDS for lipophilic molecules were comparable to those of untreated L-PGDS. Taken together, these results demonstrate that L-PGDS protected against neuronal cell death by scavenging reactive oxygen species without losing its ligand-binding function. The novel function of L-PGDS could be useful for the suppression of oxidative stress-mediated neurodegenerative diseases.]]> A Fukuhara, M Yamada, K Fujimori, Y Miyamoto, T Kusumoto, H Nakajima, T Inui 2012-01-16T14:00:57Z doi:10.1042/BJ20111889 Portland Press Limited 2012-01-16 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111881 i) is present, and that inhibition is lost when P_i is substituted with arsenate (As_i) or vanadate (V_i). To test this, liver mitochondria were isolated from wildtype and CypD-deficient (Ppif^-/-) mice and then incubated in buffer containing P_i, As_i, or V_i. MPT was induced under both energized and de-energized conditions by addition of Ca²⁺, and the resultant mitochondrial swelling measured spectrophotometrically. For pharmacological inhibition of CypD, wildtype mitochondria were pre-incubated with cyclosporine-A (CsA) prior to the addition of Ca²⁺. In energized and de-energized mitochondria, Ca²⁺ induced MPT regardless of the anion present, although the magnitude differed between P_i, As_i, and V_i. However, in all cases, pretreatment with CsA significantly inhibited MPT. Moreover, these effects were independent of mouse strain, organ type, and rodent species. Similarly, attenuation of Ca²⁺-induced MPT in the Ppif^-/- mitochondria was still observed irrespective of whether P_i, As_i, or V_i was present. We conclude that the pharmacological and genetic inhibition of CypD is still able to attenuate MPT even in the absence of P_i.]]> A M McGee, C P Baines 2012-01-11T16:38:05Z doi:10.1042/BJ20111881 Portland Press Limited 2012-01-11 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20110992 Saccharomyces cerevisiae the Oac (ScOac) is a specific anion-proton symporter, the Yarrowia lipolytica Oac (YlOac) has the added ability to transport protons, thereby mimicking an uncoupling protein (UCP). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, Tyr/Phe and Met/Leu, and we studied their role by expressing them in an Oac-null S. cerevisiae strain following site-directed mutagenesis. No phenotype could be associated to the Met/Leu substitution, whereas UCP-like activity was dependent on the presence of the Tyr normally expressed in the YlOac, i.e., Tyr-ScOac also mediated proton transport, while Phe-YlOac lost its protonophoric activity. These findings indicate that, the UCP-like activity of YlOac is determined by a single tyrosine residue.]]> L Alberto Luévano-Martínez, C Barba-Ostria, D Araiza-Olivera, N Chiquete-Félix, S Guerrero-Castillo, E Rial, D Georgellis, S Uribe-Carvajal 2012-01-11T14:08:46Z doi:10.1042/BJ20110992 Portland Press Limited 2012-01-11 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111284 o-a subunit of F₁F_o-ATP synthase (F₁F_o) is a highly hydrophobic protein with five putative transmembrane helices and it plays a central role in H⁺-translocation that is coupled with ATP synthesis/hydrolysis. Here, we show that F_o-a subunit produced by the in vitro protease-free protein synthesis system (PURE system) is integrated into a preformed F_o-a-less F₁F_o complex in the Escherichia coli membrane vesicles and in liposomes. The resulting F₁F_o has H⁺-coupled ATP synthesis/hydrolysis activity that is approximately half of that of the native F₁F_o. By using this procedure, we analyzed five mutations of F₁F_o, where the conserved residues in F_o-a subunit (N90, D112, R169, N173, and Q217) were individually replaced with alanine. All of the mutant F_o-a subunits were successfully incorporated into F₁F_o, showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant F_o-a subunits were not found in F₁F_o. A mutant N173A retained full activity and mutants D112A and Q217A weak but detectable activity. No activity was observed for mutants of R169A, as reported, and N90A. N90 is located in the middle of putative second transmembrane helix and likely to play an important role in H⁺-translocation. This work exemplifies that the PURE system provides an alternative approach when in vivo expression of membraneous components in protein complexes turns out to be difficult.]]> Y Kuruma, T Suzuki, S Ono, M Yoshida, T Ueda 2011-12-14T12:07:26Z doi:10.1042/BJ20111284 Portland Press Limited 2011-12-14 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111877 Anabaena PCC 7120 genome harbours seven genes/ORFs with homology to peroxiredoxins. One of these (all1541) was identified to encode a novel glutaredoxin (Grx) domain-containing peroxiredoxin by bioinformatic analysis. A recombinant N-terminal His-tagged All1541 protein was overexpressed in E. coli and purified. Analysis with protein alkylating agent AMS showed All1541 to form an intra-molecular disulfide bond. The All1541 protein used glutathione (GSH) more efficiently than thioredoxin (Trx) to detoxify H₂O₂. Deletion of Grx domain from All1541 resulted in loss of GSH-dependent peroxidase activity. Employing site directed mutagenesis, the cysteine residues at position 50 and 75 were identified as peroxidatic and resolving cysteine residues respectively, while both the cysteine residues within the Grx domain (position 181 and 184) were shown to be essential for GSH-dependent peroxidase activity. Based on these data, a reaction mechanism has been proposed for All1541. In vitro All1541 protein protected plasmid DNA from oxidative damage. In Anabaena PCC 7120, the all1541 was transcriptionally activated under oxidative stress. Recombinant Anabaena PCC 7120 strain over-expressing All1541 protein showed superior oxidative stress tolerance to H₂O₂ as compared to the wild-type strain. The results suggest that the glutathione dependent peroxidase All1541 plays an important role in protecting Anabaena from oxidative stress.]]> M Banerjee, A Ballal, S Kumar Apte 2011-12-08T10:28:25Z doi:10.1042/BJ20111877 Portland Press Limited 2011-12-08 BJ Plant