Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.
(25/822)
Kidney proximal tubule cells developed severe energy deficits during hypoxia/reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (DeltaPsi(m)). The energy deficit, respiratory inhibition, and loss of DeltaPsi(m) were strongly ameliorated by provision of alpha-ketoglutarate plus aspartate (alphaKG/ASP) supplements during either hypoxia or only during reoxygenation. Measurements of (13)C-labeled metabolites in [3-(13)C]aspartate-treated cells indicated the operation of anaerobic pathways of alphaKG/ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of alphaKG/ASP also mitigated the loss of DeltaPsi(m) that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F(1)F(0)-ATPase activity, had been responsible for maintenance of DeltaPsi(m) by the substrates. Thus, tubule cells subjected to hypoxia/reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and/or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain DeltaPsi(m) via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia. (+info)
Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds.
(26/822)
The first step of amino acid degradation in lactococci is a transamination, which requires an alpha-keto acid as the amino group acceptor. We have previously shown that the level of available alpha-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding alpha-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce alpha-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added alpha-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added alpha-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding alpha-ketoglutarate to improve aroma development in cheese. (+info)
Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-coAs and requirement for GTP or ATP and Mg(2+) in addition to its known hydroxylation cofactors.
(27/822)
Phytanoyl-CoA hydroxylase is a peroxisomal alpha-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs. A polyhistidine-tagged human phytanoyl-CoA hydroxylase was expressed in E. coli and subsequently purified as an active protein. The recombinant enzyme required GTP or ATP and Mg(2+), in addition to its known cofactors Fe(2+), 2-oxoglutarate, and ascorbate. The enzyme was active towards phytanoyl-CoA and 3-methylhexadecanoyl-CoA, but not towards 3-methylhexadecanoic acid. Racemic, R- and S-3-methylhexadecanoyl-CoA were equally well hydroxylated. Hydroxylation of R- and S-3-methylhexadecanoyl-CoA yielded the (2S, 3R) and (2R,3S) isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, respectively. Human phytanoyl-CoA hydroxylase did not show any activity towards 2-methyl- and 4-methyl-branched acyl-CoAs or towards long and very long straight chain acyl-CoAs, excluding a possible role for the enzyme in the formation of 2-hydroxylated and odd-numbered straight chain fatty acids, which are abundantly present in brain. In conclusion, we report the unexpected requirement for ATP or GTP and Mg(2+) of phytanoyl-CoA hydroxylase in addition to the known hydroxylation cofactors. Due to the fact that straight chain fatty acyl-CoAs are not a substrate for phytanoyl-CoA hydroxylase, 2-hydroxylation of fatty acids in brain can be allocated to a different enzyme/pathway. (+info)
Regulatory properties of glutamate dehydrogenase from Sulfolobus solfataricus.
(28/822)
The purified glutamate dehydrogenase (GDH) from Sulfolobus solfataricus showed remarkable thermostability and retained 90-95% of the initial activity after incubation at -20 degrees C, 4 degrees C, and 25 degrees C for up to 6 months. Unlike mammalian GDHs, the activity of GDH from Sulfolobus solfataricus was not significantly affected by the presence of various allosteric effectors such as ADP, GTP, and leucine. Incubation of GDH with increasing concentration of o-phthalaldehyde resulted in a progressive decrease in enzyme activity, suggesting that the o-phthalaldehyde-modified lysine or cysteine is directly involved in catalysis. The inhibition was competitive with respect to both 2-oxoglutarate (Ki = 30 microM) and NADH (Ki = 100 microM), further supporting a possibility that the o-phthalaldehyde-modified residues may be directly involved at the catalytic site. The modification of GDH by the arginine-specific dicarbonyl reagent phenylglyoxal was also examined with the view that arginine residues might play a general role in the binding of coenzyme throughout the family of pyridine nucleotide-dependent dehydrogenases. The purified GDH was inactivated in a dose-dependent manner by phenylglyoxal. Either NADH or 2-oxoglutarate did not gave any protection against the inactivation caused by a phenylglyoxal. This result indicates that GDH saturated with NADH or 2-oxoglutarate is still open to attack by phenylglyoxal. Phenylglyoxal was an uncompetitive inhibitor (Ki = 5 microM) with respect to 2-oxoglutarate and a noncompetitive inhibitor (Ki = 6 microM) with respect to NADH. The above results suggests that the phenylglyoxal-modified arginine residues are not located at the catalytic site and the inactivation of GDH by phenylglyoxal might be due to a steric hindrance or a conformational change affected by the interaction of the enzyme with its inhibitor. (+info)
Catabolism of alpha-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum: evidence for an alternative tricarboxylic acid cycle.
(29/822)
A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing alpha-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of alpha-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate alpha-[U-(14)C]ketoglutarate and [U-(14)C]glutamate at rates similar to those of wild-type B. japonicum, indicating that there was an alternative route for alpha-ketoglutarate catabolism. Because cell extracts from B. japonicum decarboxylated [U-(14)C]glutamate very slowly, the gamma-aminobutyrate shunt is unlikely to be the pathway responsible for alpha-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent alpha-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP(i). Thin-layer chromatography showed that the product of alpha-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent alpha-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for alpha-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation. (+info)
Zn2+ inhibits alpha-ketoglutarate-stimulated mitochondrial respiration and the isolated alpha-ketoglutarate dehydrogenase complex.
(30/822)
Intracellular free Zn(2+) is elevated in a variety of pathological conditions, including ischemia-reperfusion injury and Alzheimer's disease. Impairment of mitochondrial respiration is also associated with these pathological conditions. To test whether elevated Zn(2+) and impaired respiration might be linked, respiration of isolated rat liver mitochondria was measured after addition of Zn(2+). Zn(2+) inhibition (K(i)(app) = approximately 1 micrometer) was observed for respiration stimulated by alpha-ketoglutarate at concentrations well within the range of intracellular Zn(2+) reported for cultured hepatocytes. The bc(1) complex is inhibited by Zn(2+) (Link, T. A., and von Jagow, G. (1995) J. Biol. Chem. 270, 25001-25006). However, respiration stimulated by succinate (K(i)(app) = approximately 6 micrometer) was less sensitive to Zn(2+), indicating the existence of a mitochondrial target for Zn(2+) upstream from bc(1) complex. Purified pig heart alpha-ketoglutarate dehydrogenase complex was strongly inhibited by Zn(2+) (K(i)(app) = 0.37 +/- 0.05 micrometer). Glutamate dehydrogenase was more resistant (K(i)(app) = 6 micrometer), malate dehydrogenase was unaffected, and succinate dehydrogenase was stimulated by Zn(2+). Zn(2+) inhibition of alpha-ketoglutarate dehydrogenase complex required enzyme cycling and was reversed by EDTA. Reversibility was inversely related to the duration of exposure and the concentration of Zn(2+). Physiological free Zn(2+) may modulate hepatic mitochondrial respiration by reversible inhibition of the alpha-ketoglutarate dehydrogenase complex. In contrast, extreme or chronic elevation of intracellular Zn(2+) could contribute to persistent reductions in mitochondrial respiration that have been observed in Zn(2+)-rich diseased tissues. (+info)
Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose.
(31/822)
Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By (13)C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol. (mg of protein)(-1). min(-1), respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol. [mg of protein](-1). min(-1)) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol. [mg of protein](-1). min(-1)). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol. [mg of protein](-1). min(-1)) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures. (+info)
Effect of oral glucose on leucine turnover in human subjects at rest and during exercise at two levels of dietary protein.
(32/822)
The aim of this study was to determine the effect of glucose supplementation on leucine turnover during and after exercise and whether variation in the previous dietary protein content modulated this effect. Postabsorptive subjects received a primed constant [1-13C, 15N]leucine infusion for 6 h, after previous consumption of a high (1.8 g kg-1 day-1, HP, n = 16) or low (0.7 g kg-1 day-1, LP, n = 16) protein diet for 7 days. The subjects were studied at rest; during 2 h of exercise, during which half of the subjects from each dietary protocol received 0.75 g kg-1 h-1 glucose (HP + G, LP + G) and the other half received water (HP + W, LP + W); then again for 2 h of rest. Glucose supplementation suppressed leucine oxidation (P < 0.01) by 20% in subjects consuming the high protein diet (58.2 +/- 2.8 micromol kg-1 h-1, HP + G; 72.4 +/- 3.9 micromol kg-1 h-1, HP + W) but not the low protein diet (51.1 +/- 5.9 micromol kg-1 h-1, LP + G; 51.7 +/- 5.5 micromol kg-1 h-1, LP + W), with no difference in skeletal muscle branched-chain 2-oxo acid dehydrogenase (BCOADH) activity between groups. Glucose supplementation did not alter the rate of whole-body protein synthesis or breakdown. The sparing effect of glucose on leucine oxidation appears only to occur if previous protein intake was high. It was not mediated by a suppression of BCOADH fractional activity but may be due to reduced substrate availability. (+info)