Measurement of short-chain acyl-CoA dehydrogenase (SCAD) in cultured skin fibroblasts with hexanoyl-CoA as a competitive inhibitor to eliminate the contribution of medium-chain acyl-CoA dehydrogenase (original) (raw)
Related papers
Archives of Biochemistry and Biophysics, 1996
The acyl-CoA dehydrogenases (ACDs) 3 are a family The acyl-CoA dehydrogenases are a family of re-of related enzymes which catalyze the a,b-dehydrogelated enzymes which catalyze the a,b-dehydrogena-nation of acyl-CoA esters, transferring electrons to election of acyl-CoA esters, transferring electrons to tron-transferring flavoprotein [ETF, (1-6)]. Deficiencelectron-transferring flavoprotein. A cDNA for huies of these enzymes are important causes of human man short/branched chain acyl-CoA dehydrogenase disease (7, 8). Biochemical and immunological studies has recently been cloned, and it has been suggested have identified at least six distinct members of this that this enzyme represents the human homolog for enzyme family, each with a narrow substrate specificity the previously reported 2-methyl branched chain (2-5, 9, 10). Very long, long, medium, and short chain acyl-CoA dehydrogenase purified from rat liver. We acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and now report the cloning and expression of rat short/ SCAD) catalyze the first step in the b-oxidation of branched chain acyl-CoA dehydrogenase and charstraight chain fatty acids with substrate optima of acterization of its substrate specificity. The rat en-16, 16, 8, and 4 carbons, respectively (2, 5, 9, 10). zyme is more active toward longer carbon side Isovaleryl-CoA dehydrogenase (IVD) and a 2-methyl chains than its human counterpart, while the human branched chain acyl-CoA dehydrogenase (2-meBCAD) enzyme can utilize substrates with longer primary catalyze the third step in leucine and isoleucine/valine carbon chains. In addition, short/branched chain metabolism, respectively (2-4). A purified rat 2-meBacyl-CoA dehydrogenase can utilize valproyl-CoA as CAD has been shown to have 8.4% activity with vala substrate. Northern blotting of mRNA shows ubiqproyl-CoA compared with its optimum substrate (S)-2uitous tissue expression of both the rat and human methylbutyryl-CoA (11). We have recently cloned and enzyme. Further study of these enzymes will be helpcharacterized a cDNA for human short/branched chain ful in understanding structure/function relationacyl-CoA dehydrogenase (SBCAD) and have suggested ships in this gene family.
Biochimica Et Biophysica Acta (bba) - Lipids and Lipid Metabolism, 1986
In relation to the finding that human skin fibroblasts are capable of de novo ether phospholipid biosynthesis, we have studied the properties of acyl-CoA : dihydroxyacetone phosphate acyhransferase in fibroblast homogenates using a new assay method. The results indicate that the acylation of dihydroxyacetone phosphate shows an optimum at pH 5.5 with a broad shoulder of activity up to pH 6.4 and a decline in activity up to pH 8.2. At pH 5.5 the acyltransferase accepts dihydroxyacetone phosphate, but not glycerol 3-phosphate as a substrate. Fu~~~ore, the transferase activity was found to be membrane-Lund and inactivated by Triton X-100 at concentrations above 0.025% (w/v). Similar properties have been described for the enzyme as present in rat-liver and guinea-pig liver peroxisomes. These data, together with the finding that acyl-CoA : dihydroxyacetone phosphate acyltransferase is deficient in cultured skin fibroblasts from patients without peroxisomes (Zellweger syndrome), suggest that in cultured skin fibroblasts the enzyme is primarily located in peroxisomes.
Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans
Molecular Genetics and Metabolism, 2002
The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes that catalyze the a,b-dehydrogenation of acyl-CoA esters. Two homologues active in branched chain amino acid metabolism have previously been identified. We have used expression in Escherichia coli to produce a previously uncharacterized ACD-like sequence (ACAD8) and define its substrate specificity. Purified recombinant enzyme had a k cat =K m of 0.8, 0.23, and 0.04 (lM À1 s À1 ) with isobutyryl-CoA, (S) 2-methylbutyryl-CoA, and n-propionyl-CoA, respectively, as substrates. Thus, this enzyme is an isobutyryl-CoA dehydrogenase. A single patient has previously been described whose fibroblasts exhibit a specific deficit in the oxidation of valine. Amplified ACAD8 cDNA made from patient fibroblast mRNA was homozygous for a single nucleotide change (905G > A) in the ACAD8 coding region compared to the sequence from control cells. This encodes an Arg302Gln substitution in the full-length protein (position 280 in the mature protein), a position predicted by molecular modeling to be important in subunit interactions. The mutant enzyme was stable but inactive when expressed in E. coli. It was also stable and appropriately targeted to mitochondria, but inactive when expressed in mammalian cells. These data confirm further the presence of a separated ACD in humans specific to valine catabolism (isobutyryl-CoA dehydrogenase, IBDH), along with the first enzymatic and molecular confirmation of a deficiency of this enzyme in a patient. Ó
Molecular and cellular pathology of very-long-chain acyl-CoA dehydrogenase deficiency
Molecular Genetics and Metabolism, 2013
Background-Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is diagnosed in the US through newborn screening (NBS). NBS often unequivocally identifies affected individuals, but a growing number of variant patterns can represent mild disease or heterozygous carriers. Aims-To evaluate the validity of standard diagnostic procedures for VLCADD by using functional in vitro tools. Methods-We retrospectively investigated 13 patient samples referred to our laboratory because of a suspicion of VLCADD but with some uncertainty to the diagnosis. All 13 patients were suspected of having VLCADD either because of abnormal NBS or suggestive clinical findings. ACADVL genomic DNA sequencing data were available for twelve of them. Ten of the patients had an abnormal NBS suggestive of VLCADD, with three samples showing equivocal results. Three exhibited suggestive clinical findings and blood acylcarnitine profile (two of them had a normal NBS and the third one was unscreened). Assay of VLCAD activity and immunoblotting or immunohistologic staining for VLCAD were performed on fibroblasts. Prokaryotic mutagenesis and expression studies were performed for nine uncharacterized ACADVL missense mutations. Results-VLCAD activity was abnormal in fibroblast cells from 9 patients (8 identified through abnormal NBS, 1 through clinical symptoms). For these 9 patients, immunoblotting/staining showed variable presence of VLCAD; all but one had two mutated alleles. Two patients with equivocal NBS results (and a heterozygous genotype) and the two patients with normal NBS exhibited normal VLCAD activity and normal VLCAD protein on immunoblotting/staining thus ruling out VLCAD deficiency. Nine pathogenic missense mutations were characterized with prokaryotic expression studies and showed a decrease in enzyme activity and variable stability of VLCAD antigen.
Tissue specific and developmental expression of rat long-and medium-chain acyl-CoA dehydrogenases
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1993
Utilization of fatty acids for energy varies among mammalian tissues and during development due to changes in expression of enzymes of mitochondrial /3 oxidation. To discern whether two related nuclear genes are expressed similarly, the tissue distribution and developmental profile of the rat long-and medium-chain acyl-CoA dehydrogenase (LCAD and MCAD) mRNAs were compared. A 1451 base full-length LCAD eDNA from neonatal rat aorta was used to study mRNA accumulation in adult and fetal rat tissues. LCAD and MCAD mRNAs were expressed in aorta, heart, and brown fat at levels 8-40 fold greater than in liver, kidney, and duodenum. Brain, placenta, ovary, testes, and skeletal muscle showed the least mRNA. Western blots of adult tissues with anti-rat LCAD antiserum showed corresponding amounts of LCAD protein subunits. LCAD mRNA was detectable in heart, liver, kidney, and brain of fetal rats and increased with age. LCAD and MCAD mRNAs were present in brown fat in 2-10 fold greater amounts compared to other tissues from the newborn period to the end of the weaning period. The high level of expression of LCAD and MCAD mRNA in aorta, heart, and brown fat likely reflects the high energy requirements of those tissues. Differential expression of LCAD and MCAD mRNAs reflects not only inherent gene prescribed programs, but also external influences such as hormones and diet. * Corresponding author. Fax: + 1 (317) 2744471. The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number L11276. Abbreviations: LCAD, long-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; MMDH, mitochondrial malate dehydrogenase; UCP, uncoupling protein; SDS, sodium dodecyl sulfate; 2 × SSC, 0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0; kb, kilobase; bp, base pairs; HPLC, high performance liquid chromatography.
Pediatric Research, 1992
BALB/cByJ (J) mice have short-chain acyl-CoA dehydrogenase (SCAD) deficiency and an organic aciduria similar to that of human SCAD deficiency. [9,10(n)-3H]-and 115,16(n)-3H]palmitate oxidations in J mouse fibroblasts were 96 and 35% of control, respectively, consistent with an isolated SCAD defect. Acyl-CoA dehydrogenase activities were assayed in muscle and fibroblast mitochondria from BALB/cBy controls (Y) and SCADdeficient J mice. Medium-chain acyl-CoA dehydrogenase (MCAD) activities were comparable in both J and Y mice from all tissues. In the presence of MCAD antiserum, SCAD activities in J mice were undetectable in both tissues. Apparent Km and V,,, values in liver mitochondria suggested a somewhat increased affinity of MCAD for butyryl-CoA in J mice, as compared with MCAD from other species. Immunoblot studies using mitochondria revealed identical apparent SCAD molecular weight in liver, muscle, and fibroblasts from Y mice and no detectable SCAD antigen in J mice; MCAD antigen was detected in comparable amounts from both Y and J mice. Radiolabeling and immunoprecipitation studies in J mouse fibroblasts revealed no SCAD synthesis, but normal MCAD synthesis. These data argue against the existence of tissue-specific SCAD isoforms in the mouse and confirm that this mouse strain is a model for the human organic aciduria resulting from this B-oxidation defect. (Pediatr Res 31: 552-556, 1992) Abbreviations SCAD, short-chain acyl-CoA dehydrogenase MCAD, medium-chain acyl-CoA dehydrogenase J, BALB/cByJ Y, BALB/cBy control Human SCAD deficiency is an inborn error of fatty acid oxidation reported in three infants (1, 2). Two presented with metabolic acidosis and ethylmalonic aciduria and the third presented with severe skeletal muscle weakness, developmental delay, and muscle carnitine deficiency (1, 2). Detailed enzymatic
Clinical and biochemical characterization of short-chain acyl-CoA dehydrogenase deficiency
Journal of Pediatrics
Hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency is an autosomal recessive disorder characterized by episodes of ketoacidosis and a Leigh-like basal ganglia disease, without high concentrations of pyruvate and lactate in the cerebrospinal fluid. Only 4 cases of HIBCH deficiency have been reported. However, clinical-biochemical correlation in HIBCH deficiency by determining the detailed residual enzyme activities has not yet been elucidated. Here, we report a case of two Japanese siblings with HIBCH deficiency carrying a new homozygous missense mutation (c.287C N A, [p.A96D]) at the substrate-binding site. A transfection study using HIBCH expression vectors harboring wild type or 4 reported mutations, including the newly identified mutation (p.A96D, p.Y122C, p.G317E, and p.K74Lfs*13), revealed a correlation between residual HIBCH activities and the severity of the disease. All HIBCH mutants, except p.K74Lfs*13, showed residual enzyme activity and only the patient with p.K74Lfs*13 had congenital anomalies. p.G317E showed only low enzyme activity (~3%) of that of wildtype HIBCH. Although p.A96D had approximately 7 times higher enzyme activity than p.G317E, patients with p.A96D died during childhood. These findings are essential for clinical management, genetic counseling, and specific meal and concomitant drug considerations as part of the treatment for patients with HIBCH deficiency.