http://www.biochemj.org Biochemical Journal - BJ Energy RSS Feed 0264-6021 1470-8728 Biochemical Journal http://www.biochemj.org/images/bj_Name.gif http://www.biochemj.org http://www.biochemj.org/bj/444/0199/bj4440199.htm Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His₆ tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [≈180 e·s⁻¹ (electrons per s)], UV–visible signatures of oxidized and reduced states and ability to form the P_M [‘peroxy’ (but actually a ferryl/radical state)] and F (ferryl) intermediates confirm normal functioning of the histidine-tagged protein. Point mutations were introduced into subunit I of the 6H-WT strain. All mutants were screened for their ability to assemble CcO and grow on respiratory substrate. One such mutant [6H-E243D_I (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed ~50% of the 6H-WT catalytic-centre activity, consistent with the effects of the equivalent mutation in bacterial oxidases. Mutations in both the D and the H channels affect respiratory growth and these effects are discussed in terms of their putative roles in CcO mechanism.

]]> Brigitte Meunier, Amandine Maréchal and Peter R. Rich 2012-06-01 doi:10.1042/BJ20120116 c oxidase for facile purification of mutant forms]]> Portland Press Ltd. 2012-06-01 http://www.biochemj.org/bj/444/0315/bj4440315.htm Bupivacaine is a widely used anaesthetic injected locally in clinical practice for short-term neurotransmission blockade. However, persistent side effects on mitochondrial integrity have been demonstrated in muscle parts surrounding the injection site. We use the precise language of metabolic control analysis in the present study to describe in vivo consequences of bupivacaine injection on muscle energetics during contraction. We define a model system of muscle energy metabolism in rats with a sciatic nerve catheter that consists of two modules of reactions, ATP/PCr (phosphocreatine) supply and ATP/PCr demand, linked by the common intermediate PCr detected in vivo by ³¹P-MRS (magnetic resonance spectroscopy). Measured system variables were [PCr] (intermediate) and contraction (flux). We first applied regulation analysis to quantify acute effects of bupivacaine. After bupivacaine injection, contraction decreased by 15.7% and, concomitantly, [PCr] increased by 11.2%. The regulation analysis quantified that demand was in fact directly inhibited by bupivacaine (−21.3%), causing an increase in PCr. This increase in PCr indirectly reduced mitochondrial activity (−22.4%). Globally, the decrease in contractions was almost fully explained by inhibition of demand (−17.0%) without significant effect through energy supply. Finally we applied elasticity analysis to quantify chronic effects of bupivacaine iterative injections. The absence of a difference in elasticities obtained in treated rats when compared with healthy control rats clearly shows the absence of dysfunction in energetic control of muscle contraction energetics. The present study constitutes the first and direct evidence that bupivacaine myotoxicity is compromised by other factors during contraction in vivo, and illustrates the interest of modular approaches to appreciate simple rules governing bioenergetic systems when affected by drugs.

]]> Laurent M. Arsac, Karine Nouette‑Gaulain, Sylvain Miraux, Veronique Deschodt‑Arsac, Rodrigue Rossignol, Eric Thiaudiere and Philippe Diolez 2012-06-01 doi:10.1042/BJ20112011 in vivo: a modular metabolic control analysis]]> Portland Press Ltd. 2012-06-01 http://www.biochemj.org/bj/444/0333/bj4440333.htm GHB (γ-hydroxybutyrate) is both a neurotransmitter and a drug of abuse (date-rape drug). We investigated the catabolism of this compound in perfused rat livers. Using a combination of metabolomics and mass isotopomer analysis, we showed that GHB is metabolized by multiple processes, in addition to its previously reported metabolism in the citric acid cycle via oxidation to succinate. A substrate cycle operates between GHB and γ-aminobutyrate via succinic semialdehyde. Also, GHB undergoes (i) β-oxidation to glycolyl-CoA+acetyl-CoA, (ii) two parallel processes which remove C-1 or C-4 of GHB and form 3-hydroxypropionate from C-2+C-3+C-4 or from C-1+C-2+C-3 of GHB, and (iii) degradation to acetyl-CoA via 4-phosphobutyryl-CoA. The present study illustrates the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.

]]> Guo‑Fang Zhang, Sushabhan Sadhukhan, Rafael A. Ibarra, Stephanie M. Lauden, Chia‑Ying Chuang, Sophia Sushailo, Priya Chatterjee, Vernon E. Anderson, Gregory P. Tochtrop and Henri Brunengraber 2012-06-01 doi:10.1042/BJ20112046 Portland Press Ltd. 2012-06-01