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At least 1,027 records · Page 57Linked to original sources
PubMed1986

Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.

An assay for prolyl 4-hydroxylase (EC 1.14.11.2) is described which measures succinic acid produced during the decarboxylation of 2-oxoglutaric acid in the presence of poly(L-Pro-Gly-L-Pro). [1-14C]Succinic acid was separated from its precursor 2-oxo[5-14C]glutaric acid by using ion-exchange minicolumns. The contamination of succinic acid by 2-oxoglutaric acid was approx. 1%, and the recovery of succinic acid was 100%. Kinetic parameters of prolyl 4-hydroxylase measured by the assay showed good agreement with published values. Our experience indicates that the measurement of prolyl 4-hydroxylase by the production of succinic acid is especially suited to investigations involving large numbers of assays.

Carbon Radioisotopes↗
PubMed1990

A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum.

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.

Adenine Nucleotides↗
PubMed1994

Inhibition of electron flow through complex I of the mitochondrial respiratory chain of Ehrlich ascites carcinoma cells by methylglyoxal.

The effect of methylglyoxal on the oxygen consumption of Ehrlich-ascites-carcinoma (EAC)-cell mitochondria was tested by using different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-specific inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal on the oxygen consumption by these cells. The results indicate that methylglyoxal strongly inhibits ADP-stimulated alpha-oxo-glutarate and malate plus pyruvate-dependent respiration, whereas, at a much higher concentration, methylglyoxal fails to inhibit succinate-dependent respiration. Methylglyoxal also fails to inhibit respiration which is initiated by duroquinol, an artificial electron donor. Moreover, methylglyoxal cannot inhibit oxygen consumption when the NNN'N'-tetramethyl-p-phenylenediamine by-pass is used. The inhibitory effect of methylglyoxal is identical on both ADP-stimulated and uncoupler-stimulated respiration. Lactaldehyde, a catabolite of methylglyoxal, can exert a protective effect on the inhibition of EAC-cell mitochondrial respiration by methylglyoxal. We suggest that methylglyoxal possibly inhibits the electron flow through complex I of the EAC-cell mitochondrial respiratory chain.

Aldehydes↗
PubMed1996

Autoantibodies to mitochondrial 2-oxo-acid dehydrogenase complexes in localized scleroderma.

Sera from patients with localized scleroderma frequently produce cytoplasmic staining by indirect immunofluorescence, although the antigen remains to be determined. We studied the prevalence, antigen specificity and associated clinical characteristics of anti-cytoplasmic antibodies in localized scleroderma. Serum samples from 60 patients with localized scleroderma were examined by indirect immunofluorescence analysis and immunoblotting. By immunofluorescence analysis on HEp-2 cell substrate, seven of 60 (12%) patients were shown to be positive for anti-cytoplasmic antibodies. Among these, six patients with generalized morphea had anti-mitochondrial antibodies as shown by immunoblotting: they showed reactivity with the E2 component of pyruvate dehydrogenase complex (PDC), with protein X, and with the E2 component of alpha-oxo-glutarate dehydrogenase complex, while two of them showed reactivity with PDC-E1 alpha. One of these patients who was positive for anti-PDC-E1 alpha antibody showed laboratory abnormalities, suggesting the presence of primary biliary cirrhosis. The age of disease onset was significantly higher in these six patients than in those without anti-mitochondrial antibodies. Furthermore, five of them were classified into generalized morphea with multiple plaque lesions but without linear lesions (multiple plaque type). These observations suggest that major antigens for anti-cytoplasmic antibodies in patients with localized scleroderma are mitochondrial enzymes, 2-oxo-acid dehydrogenase complexes. Patients with anti-mitochondrial antibodies may comprise a unique subset of localized scleroderma designated multiple plaque type of generalized morphea of older onset.

Adolescent↗
PubMed1998

Glutamate dehydrogenase, the marker protein of Plasmodium falciparum--cloning, expression and characterization of the malarial enzyme.

The gene of an NADP+-specific glutamate dehydrogenase was cloned from Plasmodium falciparum, the causative agent of tropical malaria. Southern-blot analysis indicates a single-copy gene. The gene encodes a protein with 470 residues which has 50% of all residues identical with those of the glutamate dehydrogenases from other low eukaryotes and eubacteria. In contrast, the sequence identity with the human enzyme is marginal, which underlines the long evolutionary distance between parasite and host. The gene was overexpressed in Escherichia coli. The kinetic properties of the recombinant enzyme are in good agreement with those of the authentic enzyme. The parasite enzyme is inhibited by D-glutamate and glutarate, but not by chloroquine. Like other coenzyme-specific glutamate dehydrogenases, but in contrast to the dual-specific mammalian enzymes, the P. falciparum enzyme is not affected by GTP and ADP. The physical and chemical properties of the protein are in accordance with the cytosol being the major localization. The gene does not encode a cleavable mitochondrial presequence and the Mr of the recombinant protein and the protein isolated from the parasite are indistinguishable on SDS/PAGE. Western-blot analysis of stage-specific parasites shows that glutamate dehydrogenase is present in all intraerythrocytic stages. The signal increased continuously from rings, early trophozoites to late trophozoites and decreased slightly in the segmenter stage. Glutamate dehydrogenase, suggested to be the major source of NADPH in the parasite, is an attractive target molecule for the rational development of new antimalarial drugs.

Amino Acid Sequence↗
PubMed2003

Development of an efficient cis-trans-cis ribozyme cassette to inactivate plant genes.

Inactivation of a targeted gene is one of the main strategies used to understand their precise cellular role. In plants, apart from chemical or physical mutagenesis and random insertions of DNA elements followed by screening for a desired phenotype, the most common strategy to inhibit the expression of a given gene involves RNA silencing. This can be achieved either through antisense suppression, sense over-expression leading to co-suppression, or expression of double-stranded DNA constructs (dsRNA). The use of ribozymes to inhibit gene product accumulation has only been occasionally attempted, mainly because of the more complex genetic engineering procedure involved, although the specificity of ribozymes can be an important factor when targeting close members of a gene family. We report here the development of a new cis-acting ribozyme cassette for the production of RNAs with desired termini. Attention to many details has been brought in order to provide a powerful procedure for plant application. For example, ultrastable GNRA tetraloops were substituted for both loops II and III of cis-acting hammerhead sequences, thereby favouring folding into the catalytically active structure that results in the self-cleavage of all transcripts. We demonstrate the usefulness of this cassette by producing a ribozyme that cleaves in trans, originally embedded in the cis-acting self-cleaving cassette. The activity of the cis-trans-cis construct, was demonstrated both in vitro and in vivo, in transgenic plants with the specific cleavage of an mRNA encoding a 2-oxo-glutarate-dependant dioxygenase predominantly expressed in pistils tissues and in leaves, from the wild potato Solanum chacoense.

Journal Article↗
PubMed2001

Ultrastructural examination of the axillary skin biopsy in the diagnosis of metabolic diseases.

There is little information in the literature regarding the usefulness of ultrastructural examination of axillary skin biopsies in the evaluation of metabolic diseases. This is a retrospective clinicopathologic review of 143 patients who underwent axillary skin biopsies as part of evaluations for metabolic disease. Twenty-three (16%) had abnormalities, classified as follows: mitochondrial (n = 12), lysosomal (n = 6), increased glycogen (n = 3), nonspecific cytoplasmic inclusions (n = 2), ceroid lipofuscinosis (n = 1), and intradermal giant cells containing vacuoles and tubular inclusions (n = 1). Muscle biopsies were performed in 13 of the 23 patients; 11 showed abnormalities, including those related to mitochondria (n = 4) and other nonspecific changes (n = 7). Two patients underwent postmortem examination. Follow-up was available in 21 patients. A clinical or biochemical diagnosis was reached in 11 patients: metachromatic leukodystrophy (n = 2), electron transport chain abnormalities (n = 2), glutaric aciduria type II (n = 1), Unverricht disease (n = 1), Lennox-Gastaut syndrome (n = 1), ketotic hypoglycemia of childhood (n = 1), probable Leigh disease (n = 1), 5-methyl tetrahydrofolate homocystine methyltransferase deficiency (n = 1), and pyruvate dehydrogenase deficiency (n = 1). Of the 120 patients with negative skin biopsy results, 29 had abnormal findings on muscle (n = 27), nerve (n = 7), or brain (n = 3) biopsies. One patient had an abnormal heart biopsy result, and 3 patients underwent postmortem examinations. Follow-up was obtained in 27 of 29 patients. Diagnoses were achieved in 15 patients: electron transport chain abnormalities (n = 5), cortical dysplasia (n = 3), myoclonic epilepsy (n = 1), leukodystrophy (n = 2), Pallister-Killian mosaic syndrome (n = 1), Rett syndrome (n = 1), Landau-Kleffner syndrome (n = 1), and mitochondrial cardiomyopathy (n = 1). In conclusion, axillary skin biopsy is helpful in the evaluation of some causes of metabolic disease, but often the findings are nonspecific. A negative biopsy result does not rule out the possibility of metabolic disease, but a positive result may provide direction for further evaluation.

Adolescent↗
PubMed2001

Detection of antimitochondrial autoantibodies in immunofluorescent AMA-negative patients with primary biliary cirrhosis using recombinant autoantigens.

Antimitochondrial antibodies (AMA) are the serologic hallmark of primary biliary cirrhosis (PBC). However, depending on the clinical laboratory, from 5% to 17% of PBC patients are consistently AMA-negative, using native mitochondrial antigens and a variety of conventional assays including immunofluorescence (IMF) and enzyme-linked immunosorbent assay (ELISA). The major immunoreactive mitochondrial autoantigens are the E2 members of the 2-oxo-acid dehydrogenase complex family, including pyruvate dehydrogenase complex-E2 (PDC-E2), branched chain 2-oxo acid dehydrogenase complex-E2 (BCOADC-E2), and oxo-glutarate dehydrogenase complex-E2 (OGDC-E2); cDNAs of these proteins have now been cloned, sequenced, and their B-cell epitopes defined. In the present study, we cloned cDNAs encoding these proteins from human, not bovine, sources, and expressed the recombinant proteins in a newly developed ELISA that employs a unique Escherichia coli buffer, and compared the data with previous assays using both AMA-positive and -negative patients. Using this new assay and our criteria for positive as an optical density (OD) greater than 10 SD above the mean of control sera, the AMA-positive rate of 191 PBC sera was 94% (179 of 191) compared with 84% (161 of 191) by IMF. None of the 316 control sera were reactive. Using our recombinant assays, we focused attention on the 30 IMF-AMA-negative patients. Twenty-two of 30 (73%) of these patients were positive using this new ELISA. The group of 30 IMF-AMA-negative/ELISA-positive patients did not differ significantly from a comparable population of IMF-AMA-positive patients with respect to age, sex distribution, liver function tests, elevation of serum IgM, or pathologic stage.

Autoantibodies↗
PubMed2002

White matter disease in cerebral organic acid disorders: clinical implications and suggested pathomechanisms.

White matter abnormalities (including dys-, hypo-, demyelination and delayed myelation) are frequently found in cerebral organic acid disorders, a recently delineated subgroup of inherited organic acid disorders presenting predominantly with a neurological symptomatology. Biochemically, this disease subgroup lacks metabolic derangements, such as hypoglycemia, hyperammonemia and acidosis, and is characterized by an accumulation of organic acids that share structural similarities with the excitatory amino acid glutamate (D-2-, L-2-, 3-hydroxyglutarate, glutarate) or have been suggested as neurotransmitters/neuromodulators ( N-acetylaspartylglutamate). Evidence from in vitro and in vivo studies is growing that relevant organic acids significantly contribute to the neuropathology of these diseases via interference with glutamatergic or GABAergic neurotransmission or impairment of energy metabolism. This article provides an overview on the clinical and neuroradiological presentation of white matter disease in cerebral organic acid disorders, focusing on the suggested pathomechanistic relevance of excitotoxicity, oxidative stress, and impaired energy metabolism.

Brain Diseases, Metabolic↗
PubMed1997

Biochemistry and autoimmune response to the 2-oxoacid dehydrogenase complexes in primary biliary cirrhosis.

Pyruvate dehydrogenase complex (PDC), 2-oxo-glutarate dehydrogenase complex (OGDC), and the branched-chain 2-oxoacid dehydrogenase complex (BCOADC) constitute the 2-oxoacid dehydrogenase family of multienzyme complexes. These complexes, which are larger than ribosomes and which consist of multiple copies of E1, E2, and E3 subunits together with regulatory kinases and phosphatases and, in the case of PDC, an E3-binding protein (protein X), each play an important role in oxidative metabolism in mitochondria. Primary biliary cirrhosis (PBC) is associated with a high incidence of autoantibodies directed at mitochondrial autoantigens (the antimito-chondrial antibodies), identified as the E2 components of PDC, OGDC, and BCOADC, together with protein X and the E1 alpha and E1 beta subunits of PDC. The dominant B-cell autoepitope in PBC has been identified as the inner lipoic acid binding domain of PDC-E2, with the lipoic acid co-factor, which plays a critical role in E2 enzymatic activity, playing a role in autoantibody binding to antigen. Autoreactive CD4+ T cells specific for human PDC-E2 are also present in both the peripheral blood and liver mononuclear cell infiltrates of PBC patients. The mechanism of break-down of B-cell and T-cell self-tolerance to these ubiquitous mitochondrial antigens in such an organ-specific manner remains unclear. The apparent importance of autoreactive responses to these self-antigens does, however, raise the possibility that antigen-specific immunotherapy may offer a novel route to therapy in PBC.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1996

Clinical course, early diagnosis, treatment, and prevention of disease in glutaryl-CoA dehydrogenase deficiency.

BACKGROUND: Glutaryl-CoA dehydrogenase deficiency (GDD) is a recessively inherited neurometabolic disorder associated with encephalopathic crises and severe extrapyramidal symptoms. Treatment regimens including glucose and electrolyte infusions during acute illnesses, oral carnitine supplementation and/or a low-protein or lysine-restricted diet have been recommended, but their efficacy has been documented only on an anecdotal basis. SUBJECTS AND METHODS: We conducted a retrospective analysis of 57 patients with proven GDD-relating appearance and severity of neurological disease to age and clinical status at diagnosis, glutaric acid levels in body fluids, and different treatment regimens. RESULTS: Thirty-six patients were diagnosed after the onset of neurological disease (symptomatic group), twenty-one before (presymptomatic group). Carnitine levels were found to be reduced in all patients at diagnosis. In the symptomatic group, macrocephaly had been present around birth and was followed by rapid postnatal head growth in 70% of the children. The patients often showed symptoms such as hypotonia, irritability, and jitteriness followed by an acute encephalopathic crisis occurring on average at 12 months of age. Common neuroimaging findings included frontotemporal atrophy, subependymal pseudocysts, delayed myelination, basal ganglia atrophy, chronic subdural effusions and hematomas. In four patients the latter two findings were initially misinterpreted as resulting from child abuse. Other important misdiagnoses in older siblings who were affected and went undiagnosed include postencephalitic cerebral palsy, dystonic cerebral palsy and sudden infant death syndrome. Metabolic treatment did not convincingly improve the neurological disease, although it may have prevented further deterioration. Symptomatic treatment with baclofen or benzodiazepines was effective in reducing muscle spasms. Children in the presymptomatic group were diagnosed because of familiarity for the disease (n = 13), macrocephaly and/or additional minor neurological signs in infancy (n = 6), or acute encephalopathy, which was fully reversible after prompt treatment (n = 2). After diagnosis, all children were treated with oral carnitine, fluid infusion during intercurrent illnesses and, in addition, a diet was started in 13 of the 21 children. All 21 children except one (born prematurely at 31 weeks) have continued to develop normally up to now. Mean age at report is 6.3 years with a range from 6 months to 14.8 years. In older patients, the neuroradiological changes, present in infancy as in the symptomatic patients, became less prominent and in one girl disappeared. CONCLUSIONS: In presymptomatic children with GDD, the onset of neurological disease can be prevented by vigorous treatment of catabolic crises during illnesses together with carnitine supplementation. The importance of dietary therapy remains unclear and needs further evaluation. The potential treatability of GDD calls for increased attention to early presenting signs in order to recognize the disorder and to initiate treatment before the onset of irreversible neurological disease.

Adolescent↗
PubMed1995

Oxidative metabolism in Rett syndrome: 2. Biochemical and molecular studies.

In an attempt to identify a possible defect of mitochondrial metabolism in Rett syndrome we studied 9 girls with typical Rett syndrome using a clinical protocol designed to identify disorders of oxidative metabolism. One girl, (RO) had marked lactic acidemia. Biochemical studies on samples from these patients included leukocyte pyruvate carboxylase assay, serum biotinidase and skin fibroblast pyruvate production, pyruvate dehydrogenase, citrate synthetase and 2-oxoglutarate dehydrogenase assay. Muscle electron transport activities were studied on samples from 4 typical Rett patients including RO. Mitochondrial DNA (mtDNA) mutational analysis for the np3243 MELAS mutation, the np8993 NARP mutation, the np8344 MERFF mutation and the 4977 kb common deletion found in Kearns-Sayre syndrome and aged tissues were tested for in 1 of the muscle samples and 2 blood samples from typical Rett patients. Western blotting of electron transport complex III was performed on mitochondrial samples obtained from autopsy brain tissue in 2 Rett patients and compared to pediatric control brain samples. No abnormalities were found in blood biotinidase or pyruvate carboxylase. Western blotting of 2 Rett brain mitochondrial samples for complex III appear normal. Pyruvate consumption in medium from 8 Rett fibroblast lines grown with and without dichloroacetate (DCA) showed a normal fall in pyruvate suggesting normal pyruvate dehydrogenase activity in these cells, however the fibroblasts from patient RO had a high pyruvate production in culture. Pyruvate dehydrogenase, 2-oxo-glutarate dehydrogenase and citrate synthetase activities in 8 Rett fibroblast lines were normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗
PubMed1989

Primary biliary cirrhosis: current knowledge, perspectives, and future directions.

These perspectives from the first International Symposium on Primary Biliary Cirrhosis review recent advances and single out some areas for further enquiry. The latter include frequency and type of associated autoimmune diseases, the existence of clinical subsets of PBC, immunohistochemical analysis of lymphoid infiltrates in the liver, effects of immunosuppressive and other treatment regimens, and models for predicting the optimal time for liver transplantation. The M2 autoantigens have been identified as mitochondrial 2-oxo-acid dehydrogenase enzymes. These include pyruvate dehydrogenase (70-74 kd antigen) and branched chain 2-oxo-acid dehydrogenase and 2-oxo-acid glutaric dehydrogenase (45-52 kd antigens). Each of these enzymes has three subunits, E1 to E3. For PDH, an autoepitope has been identified as a decapeptide containing the attachment site of lipoic acid, an essential cofactor for enzyme activity. Current questions include the degree to which antibodies to PDH, and related enzymes, account for the mitochondrial reactivity defined by immunofluorescence or other procedures, the cell-surface expression of M2 autoantigens, and the significance of the occurrence of nonmitochondrial (such as centromeric) autoantibodies in PBC. The unknown T lymphocyte contribution to the autoimmune response in PBC may involve inducer and effector components. A postulated T-cell autoepitope may be presented, in association with MHC class I or class II molecules, on the surface of biliary epithelial cells. T cell lines from PBC livers removed during transplantation could provide data on the T-lymphocyte contribution to the pathogenesis of PBC.

Humans↗
PubMed1986

[HPLC detection of pathologic metabolites of melanin and melatonin metabolism in the urine in malignant melanoma of the choroid].

In 19 patients with malignant melanoma of the choroid and 11 controls 20 different metabolic parameters of melanin and melatonin metabolism in urine were examined by high-pressure liquid chromatography (HPLC). Significant differences were found in DOPA, DOPAC, MHPG, alpha-keto-glutaric acid, pseudo-uridine, serotonin, OH-tryptophan and homovanillic acid.

Choroid Neoplasms↗
PubMed1976

Jamaican vomiting sickness. Biochemical investigation of two cases.

We identified methylenecyclopropylacetic acid, a known metabolite of hypoglycin A, in the urine of two patients with Jamaican vomiting sickness. Excretion of unusual dicarboxylic acids such as 2-ethylmalonic, 2-methylsuccinic, glutaric, adipic and dicarboxylic acids with eight and 10 carbon chains were also detected in both patients. The amounts of these dicarboxylic acids were 70 to 1000 times higher than normal. These metabolites have also been identified in urine of hypoglycin-treated rats. This evidence links hypoglycin A to Jamaican vomiting sickness as its causative agent. Urinary excretion of short-chain fatty acids was also increased up to 300 times higher than normal. These results indicate that, despite their clinical and histologic similarities, the cause and biochemical mechanisms of Jamaican vomiting sickness differ distinctly from those of Reye's syndrome in which these abnormal urinary metabolites are not appreciably increased.

Animals↗
PubMed2006

Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri.

The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii) two chiral intermediates were discovered, and (iii) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by the ndhL/ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one [2Fe-2S](2+/1+) and two [4Fe-4S](2+/1+) clusters. Enamidase, a bifunctional Fe-Zn enzyme belonging to the amidohydrolase family, mediates hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to ammonia and (S)-2-formylglutarate. NADH-dependent reduction of the latter to (S)-2-(hydroxymethyl)glutarate is catalyzed by a member of the 3-hydroxyisobutyrate/phosphogluconate dehydrogenase family. A [4Fe-4S]-containing serine dehydratase-like enzyme is predicted to form 2-methyleneglutarate. After the action of the coenzyme B(12)-dependent 2-methyleneglutarate mutase and 3-methylitaconate isomerase, an aconitase and isocitrate lyase family pair of enzymes, (2R,3S)-dimethylmalate dehydratase and lyase, completes the pathway. Genes corresponding to the first three enzymes of the E. barkeri nicotinate catabolism were identified in nine Proteobacteria.

Aminohydrolases↗
PubMed1996

Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution.

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-A resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the alpha subunit and the third domain is made up entirely by the beta subunit. The N-terminal portion of the alpha subunit and the majority of the beta subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the alpha subunit. Alignment of all the known sequences for the ETF alpha subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of alpha T266, suggesting why the pathogenic mutation, alpha T266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4'-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between alpha H286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

Amino Acid Sequence↗
PubMed1998

Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II.

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length. In situ hybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.

Amino Acid Sequence↗