http://www.biochemj.org Biochemical Journal RSS feed -- BJ Metabolism 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=BJ20120618 R Tobe, M Yoo, N Fradejas, B A Carlson, S Calvo, V N Gladyshev, D L Hatfield 2012-05-17T12:46:10Z doi:10.1042/BJ20120618 Portland Press Limited 2012-05-17 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111920 K Hanzelka, L Skalniak, J Jura, S Lenzen, E Gurgul-Convey 2012-05-16T11:22:15Z doi:10.1042/BJ20111920 Portland Press Limited 2012-05-16 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120439 vitro. In this article we analyze the data showing the dependence of this parameter upon muscle contractile state. Most remarkable is the effect of recently described calcium- independent contraction of permeabilized muscle fibers induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous calcium-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibers that resulting in a disorganization of mitochondrial arrangement leading to significant increase of affinity for ADP. These data show that calcium-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore, use of their inhibitors is strongly advised at the step of permeabilization with careful washing of fibers or cells afterwards. Possible physiologically relevant relationship between calcium-regulated, ATP-dependent contraction and mitochondrial functional parameters is also discussed.]]> A V. Kuznetsov, R Guzun, F Boucher, R Bagur, T Kaambre, V A Saks 2012-05-03T10:36:08Z doi:10.1042/BJ20120439 Portland Press Limited 2012-05-03 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120232 N-glycosylation has been linked to increased basal cell surface levels while N-glycosylation-deficient GLUT4 was found to be quickly degraded. As recycling and degradation of GLUT4 are positively correlated, we hypothesized that incorrect N-glycosylation of GLUT4 might reduce its intracellular retention resulting in an increased cell surface recycling, in increased basal cell surface levels, and in enhanced GLUT4 degradation. Here, we have studied N-glycosylation-deficient GLUT4 in detail in 3T3-L1 preadipocytes, 3T3-L1 adipocytes, and L6 myoblasts. We have found no alterations in retention, insulin response, internalization, or glucose transport activity. Degradation of the mutant molecule was increased, though once present at the cell surface, its degradation was identical to that of wild-type GLUT4. Our findings indicate that N-glycosylation is important for efficient trafficking of GLUT4 to its proper compartments, but once the transporter has arrived there, N-glycosylation plays no further major role in its intracellular traffic, nor in its functional activity.]]> N Zaarour, M Berenguer, Y Le Marchand-Brustel, R Govers 2012-04-30T14:17:26Z doi:10.1042/BJ20120232 Portland Press Limited 2012-04-30 BJ Cell http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120528 Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires’ disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive in these environments. The molecular mechanisms by which this bacterium detoxifies these chemicals remain poorly understood. In particular, the expression and functions of xenobiotic-metabolizing enzymes (XME) that could contribute to chemical detoxification in L. pneumophila have been poorly documented at the molecular and functional levels. We report here the identification and biochemical and functional characterization of a unique acetyltransferase that metabolizes aromatic amine chemicals in three characterized clinical strains of L. pneumophila (Paris, Lens, and Philadelphia). Strain-specific sequence variations in this enzyme, an atypical member of the arylamine N-acetyltransferase family (EC 2.3.1.5), produce enzymatic variants with different structural and catalytic properties. Functional inactivation and complementation experiments showed that this acetyltransferase allows L. pneumophila to detoxify aromatic amine chemicals and grow in their presence. Our study provides a new enzymatic mechanism by which the opportunistic pathogen L. pneumophila biotransforms and detoxifies toxic aromatic chemicals. These data also emphasize the role of xenobiotic-metabolizing enzymes in the environmental adaptation of certain prokaryotes.]]> X Kubiak, D Dervins-Ravault, B Pluvinage, A Chaffotte, L Gomez-Valero, J Dairou, F Busi, J Dupret, C Buchrieser, F Rodrigues-Lima 2012-04-30T13:36:25Z doi:10.1042/BJ20120528 Portland Press Limited 2012-04-30 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120173 2 allosteric site are formed by opposing N- and C-termini of subunits oriented antiparallel in a dimer. On the contrary, we show here that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in phosphofructokinase-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable, fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.]]> O H. Martínez-Costa, V Sánchez, A Lázaro, E D. Hernández, K Tornheim, J J. Aragón 2012-04-25T09:49:43Z doi:10.1042/BJ20120173 Portland Press Limited 2012-04-25 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112142 succination of proteins, increases in adipocytes grown in high glucose medium and in adipose tissues of type 2 diabetic mice. However, the metabolic mechanisms leading to increased fumarate and succination of protein in the adipocyte are unknown. Treatment of 3T3 cells with high glucose (30 mM vs. 5 mM) caused a significant increase in cellular ATP/ADP, NADH/NAD⁺, and the mitochondrial membrane potential. There was also a significant increase in cellular fumarate concentration and succination of proteins, which may be attributed to the increase in NADH/NAD⁺ and subsequent inhibition of Krebs cycle NAD⁺-dependent dehydrogenases. Chemical uncouplers, which dissipated the mitochondrial membrane potential and reduced the NADH/NAD­⁺ ratio, also decreased fumarate concentration and protein succination. High glucose plus metformin, an inhibitor of Complex I in the electron transport chain, caused an increase in fumarate and succination of protein. Thus, excess fuel supply (glucotoxicity) appears to create a pseudohypoxic environment (high NADH/NAD⁺ without hypoxia), which drives the increase in succination of protein. We propose that increased succination of proteins is an early marker of glucotoxicity and mitochondrial stress in adipose tissue in diabetes.]]> N Frizzell, S A Thomas, J A Carson, J W Baynes 2012-04-24T10:00:12Z doi:10.1042/BJ20112142 Portland Press Limited 2012-04-24 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112225 Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV-infected) patients, and (ii) known to preferentially colonize tumor tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a thymidine phosphorylase (TP) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyzes the phosphorolysis of thymidine (K_m = 473 µM) and deoxyuridine (K_m = 578 µM), it prefers uridine (K_m = 92 µM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the nucleoside phosphorylase (NP)-II class pyrimidine nucleoside phosphorylases (PyNP), and is distinct from the NP-II class TP and NP-I class uridine phosphorylases (UP). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.]]> J Vande Voorde, F Gago, K Vrancken, S Liekens, J Balzarini 2012-04-04T14:17:03Z doi:10.1042/BJ20112225 Portland Press Limited 2012-04-04 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112200 Escherichia coli with exogenously added heme at elevated temperature in the presence of a relatively high concentration of sodium chloride. Kinetic analysis of iron oxidation by E. coli BFR preparations containing varying amounts of heme revealed that heme bound to BFR decreases the rate of iron oxidation at the dinuclear iron ferroxidase sites but increases the rate of iron core formation. Similar kinetic analysis of BFR reconstituted with cobalt-heme revealed that this heme derivative has no influence on the rate of iron core formation. These observations argue that heme bound to E. coli BFR accelerates iron core formation by an electron transfer-based mechanism.]]> S G. Wong, R Abdulqadir, N E Le Brun, G R Moore, A Grant Mauk 2012-03-30T10:09:09Z doi:10.1042/BJ20112200 Portland Press Limited 2012-03-30 BJ Structure http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120294 A R Diers, K A Broniowska, C Chang, N Hogg 2012-03-30T09:49:50Z doi:10.1042/BJ20120294 Portland Press Limited 2012-03-30 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120197 Shewanella oneidensis MR-1 identifies three low-spin His/His coordinated c-hemes and a single high-spin c-heme with His/H₂O coordination lying adjacent to the quinol binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (ca. -240 mV) and low-spin (ca. -110, -190 and -265 mV) hemes that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (E_m -80 mV) in the presence of NADH (E_m -320 mV) and an NADH:menadione oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H₂ oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-heme cofactor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc₃. The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.]]> S J Marritt, T G Lowe, J Bye, D G G McMillan, L Shi, J Fredrickson, J Zachara, D J Richardson, M R Cheesman, L J C Jeuken, J N Butt 2012-03-29T14:13:17Z doi:10.1042/BJ20120197 Portland Press Limited 2012-03-29 BJ Energy http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20111890 X Dong, X Fu, S Fan, P Guo, D Su, J Dong 2012-03-28T10:46:12Z doi:10.1042/BJ20111890 Portland Press Limited 2012-03-28 BJ Gene http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20120163 (i) Augmented flux through the TCA cycle, as evidenced by larger incorporation of ¹³C into the cycle (anaplerosis) and increased generation of ¹³C isotopomers of citrate, glutamate and aspartate (cataplerosis); (ii) Lowering of hepatic [Pi] and elevated [ATP], denoting greater phosphorylation potential and energy state; (iii) Stimulation of glycogen synthesis from glucose but inhibition of glycogen synthesis from 3-carbon precursors; (iv) Increased synthesis of N-acetlylglutamate and consequently augmented ureagenesis; (v) Increased synthesis of glutamine, alanine, serine and glycine; and (vi) Increased production and outflow of lactate. The current study provides a deeper insight into the hepatic actions of GKAs and uncovers the potential benefits and risks of GKA for treatment of diabetes. GKA improved hepatic bioenergetics, ureagenesis and glycogenesis but decreased gluconeogenesis with a potential risk of lactic acidosis and fatty liver.]]> I Nissim, O Horyn, I Nissim, Y Daikhin, S L. Wehrli, M Yudkoff, F M. Matschinsky 2012-03-26T13:56:40Z doi:10.1042/BJ20120163 Portland Press Limited 2012-03-26 BJ Metabolism http://www.biochemj.org/bj/imps/refer.htm?MSID=BJ20112079 S Geula, D Ben-Hail, V Shoshan-Barmatz 2012-03-08T12:29:12Z doi:10.1042/BJ20112079 Portland Press Limited 2012-03-08 BJ Structure