PubMed Health⌕ Search

Biomedical subjects

F E Frerman

Publications and source records attributed to F E Frerman.

At least 37 records · Page 2Linked to original sources
PubMed1994

Mutations and polymorphisms of the gene encoding the beta-subunit of the electron transfer flavoprotein in three patients with glutaric acidemia type II.

Electron transfer flavoprotein (ETF) is a heterodimeric enzyme composed of an alpha-subunit and a beta-subunit and contains a single equivalent of FAD per dimer. ETF deficiency can be demonstrated in individuals affected by a severe metabolic disorder, glutaric acidemia type II (GAII). In this study, we have investigated for the first time the molecular basis of beta-ETF deficiency in three GAII patients: two Japanese brothers, P411 and P412, and a third unrelated patient, P485. Molecular analysis of the beta-ETF gene in P411 and P412 demonstrated that both these patients are compound heterozygotes. One allele is carrying a G to A transition at nucleotide 518, causing a missense mutation at codon 164. This point mutation is maternally derived and is not detected in 42 unrelated controls. The other allele carries a G to C transversion at the first nucleotide of the intron donor site, downstream of an exon that is skipped during the splicing event. The sequence analysis of the beta-ETF coding sequence in P485 showed only a C to T transition at nucleotide 488 that causes a Thr154 to Met substitution and the elimination of a HgaI restriction site. HgaI restriction analysis on 63 unrelated controls' genomic DNA demonstrated that the C488T transition identifies a polymorphic site. Finally, transfection of wild-type beta-ETF cDNA into P411 fibroblasts suggests that wild-type beta-ETF cDNA complements the genetic defect and restores the beta-oxidation flux to normal levels.

Amino Acid Metabolism, Inborn Errors↗
PubMed1993

Cloning, sequencing, and expression of the genes encoding subunits of Paracoccus denitrificans electron transfer flavoprotein.

The genes encoding the two subunits of Paracoccus denitrificans electron transfer flavoprotein (ETF) were identified by screening a genomic library constructed in pBluescript II SK+ with probes generated by amplification of genomic sequences by the polymerase chain reaction. Primers for the polymerase chain reaction were designed based on peptide sequences from purified Paracoccus ETF subunits. The genes are arranged in tandem in the genomic DNA with the deoxyadenylic acid residue in the TGA termination codon of the small subunit providing the deoxyadenylic acid residue for the ATG initiating codon of the large subunit. The deduced amino acid sequences of the ETF subunits exhibits extensive sequence identity with the human ETF subunits. The Paracoccus ETF is expressed from the pBluescript vector in Escherichia coli, yielding 30 mg of purified, catalytically active protein per liter of culture.

Amino Acid Sequence↗
PubMed1992

Redox properties of electron-transfer flavoprotein ubiquinone oxidoreductase as determined by EPR-spectroelectrochemistry.

We have determined the formal potential values for each electron transfer to electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), in order to further characterize the thermodynamics of electron transport from various acyl-CoA thioesters to the mitochondrial ubiquinone pool. ETF-QO contains one [4Fe-4S]2+,1+ cluster and one FAD prosthetic group. A preliminary visible-spectroelectrochemical titration showed that the two redox centers were reduced almost simultaneously. Since the visible spectra of the chromophores overlap, it was not possible to resolve the formal potential value for each electron transfer to the protein using this method. Accordingly, an EPR-spectroelectrochemical cell was designed so that each formal potential value could be resolved by EPR quantitation of the flavin semiquinone and the reduced iron-sulfur cluster during the titration. The formal potential values for electron transfer to ETF-ubiquinone oxidoreductase at pH 7.5 and 4 degrees C were E1 degrees' = +0.028 V and E2 degrees' = -0.006 V for the first and second electron transfers, respectively, to the FAD and E degrees' = +0.047 V for the iron-sulfur cluster. The thermodynamics of electron transport from the acyl-CoA substrates of beta-oxidation to the mitochondrial electron transport chain have been fully resolved with completion of this work. The results are discussed in terms of their significance to the overall electron transport process from beta-oxidation.

Acyl Coenzyme A↗
PubMed1992

Structural and redox relationships between Paracoccus denitrificans, porcine and human electron-transferring flavoproteins.

Electron-transferring flavoprotein (ETF) was purified from the bacterium Paracoccus denitrificans and the structural and redox relationships to the porcine and human ETFs were investigated. The three proteins have essentially identical subunit masses and the alpha-helix content of the bacterial and porcine ETFs are very similar, indicating global structural similarity. An anti-(porcine ETF) polyclonal antibody that crossreacts with the human large and small subunits also crossreacts strongly with the large subunit of Paracoccus ETF. However, crossreactivity with the small subunit is very weak. Nonetheless, an amino-terminal peptide and four internal peptides of the small bacterial subunit show extensive sequence identity with the human small subunit. Local similarities in environment are also indicated by the intrinsic tryptophan fluorescence emission spectra of porcine and Paracoccus ETFs. Although the visible spectra of porcine and Paracoccus ETFs are virtually identical, flavin fluorescence in the bacterial protein is only 15% that of the mammalian protein. Further, the circular dichroic spectrum of the flavin in the bacterial protein is significantly more intense, suggesting that the microenvironment of the isoalloxazine ring is different in the two proteins. Enzymatic or photochemical reduction of Paracoccus ETF rapidly yields an anionic semiquinone; formation of the fully reduced flavin in the bacterial ETF is very slow. The spacing of the oxidation-reduction potentials of the flavin couples in the bacterial ETF is essentially identical to that in procine ETF as judged from the disproportionation equilibrium of the bacterial ETF flavin semiquinone. Together, the enzymatic reduction and disproportionation equilibria suggest that the flavin potentials of the two ETFs must be very close. The data indicate that the structural properties of the bacterial and mammalian proteins and the thermodynamic properties of the flavin prosthetic group of the proteins are very similar.

Amino Acid Sequence↗
PubMed1991

Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate.

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.

Acrylamide↗
PubMed1990

Glutaric acidemia type II: heterogeneity of clinical and biochemical phenotypes.

We have examined 23 fibroblast lines from patients with neonatal and late onset glutaric acidemia type II and fibroblasts from four parents of these patients. Fifteen of these patients are previously unreported. Results of these investigations show deficiency of electron transfer flavoprotein or electron transfer flavoprotein-ubiquinone oxidoreductase activity in all of the patients' fibroblasts. Immunoblots indicate that the steady state levels of the antigens is very low or undetectable in most of the neonatal onset patients; however, cross-reacting antigen without electron transfer activity is observed in several glutaric acidemia type II fibroblast lines. Assay of parental lines confirm the autosomal transmission of deficiencies of proteins. Of particular interest is the clinical heterogeneity among these patients. Patients may present with an extrapyramidal movement disorder as observed in glutaric aciduria type I, without the typical organic aciduria typical of glutaric acidemia type II even in the presence of severe enzyme deficiency, or with renal cystic dysplasia accompanying electron transfer flavoprotein deficiency. Renal cystic dysplasia had previously been reported only in patients with electron transfer flavoprotein-ubiquinone oxidoreductase deficiency.

Cell Line↗
PubMed1990

Electron transfer flavoprotein: ubiquinone oxidoreductase (ETF:QO) deficiency in an adult.

A 19-year-old woman with mild myopathic symptoms from age 6 and fasting intolerance presented with a Reye-like syndrome and a myopathy. Investigations disclosed a lipid storage myopathy, type II glutaric acidemia, and carnitine deficiency in skeletal muscle. Riboflavin and carnitine treatment corrected the metabolic abnormalities and she improved clinically. She later died from pulmonary complications secondary to aspiration. Subsequent studies established electron transfer flavoprotein: ubiquinone oxidoreductase (ETF:QO) deficiency (fibroblast ETF:QO activity was 2.9 mU/mg, normal range is 14.1 +/- 3.8 mU/mg) as the cause of her illness. This is the first documented case of ETF:QO diagnosed in an adult.

Acidosis↗
PubMed1990

Glutaric aciduria type II: autopsy study of a case with electron-transferring flavoprotein dehydrogenase deficiency.

An autopsy study of glutaric aciduria type II in a 62-day-old Japanese boy is presented. The diagnosis was made by analysis of organic acids in the urine. Immunoblot analysis of liver homogenate confirmed the diagnosis, revealing absence of electron-transferring flavoprotein dehydrogenase. The major findings were fatty changes of variable degree in many organs and tissues, the most severe being found in cardiac myocytes, hepatocytes, renal tubular epithelium, and skeletal muscle fibers. Other pertinent findings included multicystic and dysplastic kidney, pulmonary alveolar proteinosis, and spongiosis and gliosis of the spinal cord. The thymus was markedly depleted, and lymphocytes in the lymph nodes were mainly B cells. Although some of these changes may have been secondary to the sepsis and immunosuppression complicating 2 months of intensive care, the abnormal organic acid metabolism with severe acidosis may have been a significant contributing factor.

Acidosis↗