Conservation of central nervous system glutaryl-coenzyme A dehydrogenase in fruit-eating bats with glutaric aciduria and deficient hepatic glutaryl-coenzyme A dehydrogenase

The adult fruit-eating bat, Rousettus aegypticus, excretes massive amounts of glutaric acid in the urine (20-70 mumol/mg creatinine) comparable to those of humans affected with the inherited metabolic disorder, glutaric aciduria type I. Glutaric acid was quantified by sequential liquid partition chromatography and gas chromatography. Oral loading with the amino acid precursors of glutaric acid, L-lysine and L-tryptophan, resulted in significant increases in glutaric acid excretion above the base-line values. Glutaryl-CoA dehydrogenase activity was assayed in adult bat tissues and compared with the same tissues in the rat using methods of 14CO2 evolution from 1,5-[14C]glutaryl-CoA. A severe deficiency of glutaryl-CoA dehydrogenase activity was found in the bat liver and kidney, whereas brain and spinal cord levels were similar to those in the rat. Reverse phase high performance liquid chromatography analysis of the metabolites in the assay mixture showed negligible hydrolysis of [14C]glutaryl-CoA to free [14C]glutaric acid and complete conversion of the product [14C]crotonyl-CoA to 3-hydroxy[14C]butyryl-CoA. The adult bat, with its huge glutaric acid excretion and deficient liver glutaryl-CoA dehydrogenase, metabolically mimics patients affected with glutaric aciduria type I. The bat does not, however, display the neurologic manifestations seen in patients. This may be explained by conservation of glutaryl-CoA dehydrogenase activity in the central nervous system of the bat.     

Conversion of a decarboxylating to a non-decarboxylating glutaryl-coenzyme A dehydrogenase by site-directed mutagenesis

Glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are acyl-CoA dehydrogenases, which usually dehydrogenate and decarboxylate the substrate to crotonyl-CoA. In some anaerobic bacteria, non-decarboxylating GDHs exist that release glutaconyl-CoA (2,3-dehydroglutaryl-CoA) without decarboxylation. The differing mechanisms of decarboxylating and non-decarboxylating GDHs were investigated by site-directed mutagenesis of the gene coding for the crotonyl-CoA-forming GDH from Geobacter metallireducens. Exchange of single amino acids involved in substrate carboxylate binding impaired the decarboxylation step, resulting in relative glutaconyl-CoA:crotonyl-CoA formation rates of 1:1 (S97A) or 13:1 (Y370A). The total amount of glutaconyl-CoA formed was maximal in the Y370V+S97A double mutant. The results obtained indicate that an invariant deprotonated Tyr plays a crucial role for optimizing the leaving group potential of CO(2) in decarboxylating GDHs.     

Glucose-6-phosphate dehydrogenase deficiency: a novel role for thyroxine in peroxide-induced hemolysis

G-6-PD deficient erythrocytes undergo hemolysis during exposure to hydrogen peroxide and thyroxine. Normal erythrocytes do not show this effect unless incubated in the absence of glucose. The inability of G-6-PD deficient cells to detoxify hydrogen peroxide apparently exposes them to the cytotoxic actions of thyroxine. It is suggested that the hemolysis and anemia associated with this genetic disorder is a consequence of the actions of both peroxide and thyroxine on erythrocyte membranes.     

The purification and characterization of glutaryl-coenzyme A dehydrogenase from porcine and human liver

Glutaryl-CoA dehydrogenase, a multifunctional enzyme responsible for dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA, has been purified 1,680-fold from porcine liver mitochondria. The purified porcine enzyme has a subunit molecular weight of 47,800 and a native molecular weight of 190,500. Porcine glutaryl-CoA dehydrogenase catalyzed the conversion of [1,5-14C]glutaryl-CoA to [14C] crotonyl-CoA and 14CO2 in a 1:1:1 ratio. The porcine enzyme has Km values for electron transfer flavoprotein and glutaryl-CoA of 1.1 and 3.3 microM, respectively, and turnover numbers of 860 mol of electron transfer flavoprotein/min/mol of glutaryl-CoA dehydrogenase and 327 mol of glutaryl-CoA/min/mol of glutaryl-CoA dehydrogenase. Human glutaryl-CoA dehydrogenase has been purified 1,278-fold from human liver mitochondria. The purified human enzyme has a subunit molecular weight of 58,800 and a native molecular weight of 256,000. Human glutaryl-CoA dehydrogenase showed a reaction of only partial identity when compared to porcine glutaryl-CoA dehydrogenase by Ouchterlony double immunodiffusion analysis using antiserum raised against and monospecific for porcine glutaryl-CoA dehydrogenase.     

Glucose-6-phosphate dehydrogenase deficiency: a new hypothesis for an old disease

Summary

We have previously shown insulinoma (HIT-T15 and RINm5F) cells in culture to be very sensitive, in comparison with a reference cell line (J-774), to the oxidative stress that is created when alloxan reacts extracellularly with reducing agents, forming superoxide and hydrogen peroxide. The toxic effects are prevented by catalase added to the medium, suggesting that alloxan does not need to be taken up in order to affect cells. Rather, alloxan seems to exert its action through extracellular formation of hydrogen peroxide that influences the stability of the cells' lysosomes following diffusion into them. To further analyse the mechanisms in operation, we studied the influence of induced autophagocytosis on the sensitivity to ensuing oxidative stress. Starvation for 60–120 min in PBS at 37°C markedly enhanced autophagocytosis and, in parallel, increased the cytotoxic effect and lysosomal vulnerability of ensuing exposure to hydrogen peroxide, while not significantly changing the antioxidative status or the energy balance. Autophagocytosis increased the size of the intralysosomal pool of reactive, low-molecular-weight, iron, probably by degradation of metallo-proteins, as shown by autometallography and HPLC demonstration of desferrioxamine-reactive intracellular iron. Moreover, exposure to the iron-chelator desferrioxamine before treatment with hydrogen peroxide prevented lysosomal destabilization and cellular death of both starved and control cells, further proving the importance of intralysosomal iron for the response to oxidative stress. We hypothesize that β-cells which, like insulinoma cells, have a weak antioxidative defence system under conditions of enhanced general autophagocytosis, or crinophagy, might become vulnerable to even low, or moderate, oxidative stress.     

Hamster sperm capacitation: role of pyruvate dehydrogenase A and dihydrolipoamide dehydrogenase

Recently, we demonstrated that pyruvate dehydrogenase A2 (PDHA2) is tyrosine phosphorylated in capacitated hamster spermatozoa. In this report, using bromopyruvate (BP), an inhibitor of PDHA, we demonstrated that hamster sperm hyperactivation was blocked regardless of whether PDHA was inhibited prior to or after the onset of hyperactivation, but the acrosome reaction was blocked only if PDHA was inhibited prior to the onset of the acrosome reaction. Further, inhibition of PDHA activity did not inhibit capacitation-associated protein tyrosine phosphorylation observed in hamster spermatozoa. It is demonstrated that the essentiality of PDHA for sperm capacitation is probably dependent on its ability to generate effectors of capacitation such as reactive oxygen species (ROS) and cAMP, which are significantly decreased in the presence of BP. MICA (5-methoxyindole-2-carboxylic acid, a specific inhibitor of dihydrolipoamide dehydrogenase [DLD]), another component of the pyruvate dehydrogenase complex (PDHc), also significantly inhibited ROS generation and cAMP levels thus implying that these enzymes of the PDHc are required for ROS and cAMP generation. Furthermore, dibutryl cyclic adenosine monophosphate could significantly reverse the inhibition of hyperactivation observed in the presence of BP and inhibition of acrosome reaction observed in the presence of BP or MICA. The calcium ionophore, A23187, could also significantly reverse the inhibitory effect of BP and MICA on sperm acrosome reaction. These results establish that PDHA is required for hamster sperm hyperactivation and acrosome reaction, and DLD is required for hamster acrosome reaction. This study also provides evidence that ROS, cAMP, and calcium are involved downstream to PDHA.     

Bioenergetics of the formyl-methanofuran dehydrogenase and heterodisulfide reductase reactions in Methanothermobacter thermautotrophicus.

The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H2-dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( pH2). Formyl-methanofuran synthesis is an endergonic reaction (DeltaG degrees ' = +16 kJ.mol-1). By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (DeltaG') were found to range between +10 and +35 kJ.mol-1 depending on the pH2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions. Heterodisulfide reduction (DeltaG degrees ' = -40 kJ.mol-1) was associated with free energy changes as high as -55 to -80 kJ.mol-1. The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at pH2<0.12 bar, whilst the number was four for cells grown above that concentration. The shift occurred within a narrow pH2 span around 0.12 bar. The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM-S-S-CoB reduction and proton pumping in response to the environmental hydrogen concentrations.     

Genetic basis for correction of very-long-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy

Very-long-chain acyl–coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid β-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in β-oxidation–deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 μM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and exemplifies the integration of molecular information in a therapeutic strategy.     

Glucose 6-phosphate dehydrogenase deficiency: a protection against malaria and a risk for hemolytic accidents

Glucose 6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway, which in the RBC leads to the formation of NADPH, essential to prevent the cell from an oxidative stress. Worldwide, more than 400 million people (90% being males) are affected by G6PD deficiency, in regions that are, or have been, endemic for malaria and in populations originating from these regions. RBCs with low G6PD activity offer a hostile environment to parasite growth and thus an advantage to G6PD deficiency carriers. The counterpart of this protective effect is an increased susceptibility to oxidants such as some foods (fava beans), drugs (anti-malarial or sulphonamides), or various chemicals. In the case of G6PD deficiency, the hypothesis of a convergent evolution between parasite, protecting mutation, and cultural traditions (food, skin painting...) has been proposed. Near to 150 different G6PD variants have been described, which are classified into four types, according to their clinical effects. Several variants, such as the G6PD A- or the Mediterranean variant, reach the polymorphism level in endemic regions. The recent determination of the three-dimensional structure of this enzyme allows one to explain now the mechanisms of the disorders in terms of structure-function relationship.     

Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans.

Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.     

ATP synthesis in lipoamide dehydrogenase deficiency

Lipoamide dehydrogenase deficiency is an inborn error of several metabolic pathways, including pyruvate metabolism, Krebs cycle, and branched-chain amino acid degradation. The clinical course is variable, ranging from infantile neurodegenerative disease to recurrent episodes of liver failure or myoglobinuria starting later in life. In contrast, residual enzymatic activity in muscle tissue spans over a narrow range. Despite the recent elucidation of the underlying molecular pathology in most patients, relationships between the genotype and the biochemical and clinical phenotype remain unclear. In order to find a suitable assay for the prediction of clinical outcome and assessment of treatment, we have evaluated enzymatic activities and energetic states in fibroblasts from lipoamide dehydrogenase-deficient patients representing three different phenotypes and genotypes. Direct relationships between clinical parameters such as age of onset and disease severity and biochemical characteristics, including lipoamide dehydrogenase activity, pyruvate dehydrogenase complex activity, and ATP production ratio in fibroblasts, were identified. Clinical parameters were not reflected by lactate/pyruvate ratio. ATP production rate was in direct relationship with the severity of the neurological involvement; the patient with reduced ATP synthesis to 30% of the control mean had a severe neurodegenerative disease, whereas ATP synthesis values above 45% were associated with a more favorable course. Incubation of the patients' fibroblasts with dichloroacetate coupled with thiamin resulted in slight but significant improvement of the cell energetic state.     

Partial deficiency of red cell 6-phosphogluconate dehydrogenase: A family study

Summary A family with partial deficiency of erythrocytic 6PGD is described. Biochemical and electrophoretic analysis suggest that the partial deficiency is due to a silent PGD ⁰ allele.Chromosomal analysis and assay of closely linked markers do not reveal a grossly detectable deletion.Abbreviations used in this paper G6PD glucose-6-phosphate dehydrogenase - GSSGR glutathione reductase - PK pyruvate kinase - GSH glutathione - PGM phosphoglucomutase     

Glutamate dehydrogenase deficiency in spinocerebellar degenerations

Glutamate dehydrogenase (GDH) activity in leukocytes and platelets in spinocerebellar degenerations (SCD) was determined. In the same subject, GDH activity was higher and more reproducible in platelets than in leukocytes. GDH was decreased significantly in olivopontocerebellar atrophy (OPCA) (Ca. 30% decrease). Pyruvate dehydrogenase (PDH) in platelets showed non specific decreased activity in SCD and amyotropic lateral sclerosis. Energy metabolism in cerebellum may be diminished in some types of ataxia, and glutaminergic neurons may be more affected in OPCA than in other SCD.     

Cardiac fibrogenesis in magnesium deficiency: a role for circulating angiotensin II and aldosterone

Mechanisms underlying cardiac fibrogenesis in magnesium deficiency are unclear. It was reported earlier from this laboratory that serum from magnesium-deficient rats has a more pronounced stimulatory effect on cell proliferation, net collagen production, and superoxide generation in adult rat cardiac fibroblasts than serum from rats on the control diet. The profibrotic serum factors were, however, not identified. This study tested the hypothesis that circulating angiotensin II may modulate cardiac fibroblast activity in hypomagnesemic rats. Male Sprague-Dawley rats were pair-fed a magnesium-deficient (0.0008% Mg) or -sufficient (0.05%) diet for 6 days, and the effects of serum from these rats on [3H]thymidine and [3H]proline incorporation into cardiac fibroblasts from young adult rats were evaluated in the presence of losartan, an angiotensin II type 1 (AT1) receptor antagonist, and spironolactone, an aldosterone antagonist. Losartan and spironolactone markedly attenuated the stimulatory effects in vitro of serum from the magnesium-deficient and control groups, but the inhibitory effects were considerably higher in cells exposed to serum from magnesium-deficient animals. Circulating and cardiac tissue levels of angiotensin II were significantly elevated in magnesium-deficient animals (67.6% and 93.1%, respectively, vs. control). Plasma renin activity was 61.9% higher in magnesium-deficient rats, but serum angiotensin-converting enzyme activity was comparable in the two groups. Furthermore, preliminary experiments in vivo using enalapril supported a role for angiotensin II in magnesium deficiency. There was no significant difference between the groups in serum aldosterone levels. The findings suggest that circulating angiotensin II and aldosterone may stimulate fibroblast activity and contribute to a fibrogenic response in the heart in magnesium deficiency.     

Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation in humans. To better understand the pathogenesis of this disease, we developed a mouse model for MCAD deficiency (MCAD^−/−) by gene targeting in embryonic stem (ES) cells. The MCAD^−/− mice developed an organic aciduria and fatty liver, and showed profound cold intolerance at 4 °C with prior fasting. The sporadic cardiac lesions seen in MCAD^−/− mice have not been reported in human MCAD patients. There was significant neonatal mortality of MCAD^−/− pups demonstrating similarities to patterns of clinical episodes and mortality in MCAD-deficient patients. The MCAD-deficient mouse reproduced important aspects of human MCAD deficiency and is a valuable model for further analysis of the roles of fatty acid oxidation and pathogenesis of human diseases involving fatty acid oxidation.     

Glucose-6-phosphate dehydrogenase deficiency in Spain

Examination of 2520 male subjects in Spain by the Dye Reduction Test revealed five cases of Glucose-6-phosphate dehydrogenase deficiency, all originating from the east coast of Spain and the Balearic Islands. In these areas the enzyme deficiency seems to occur focally at low frequencies near 1%.Qui de fetge va que no passi pel favar! Who suffers from the liver should not pass through a fava field!(Direktor: Prof. Dr. H. Hungerland)