Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.3.99.3 (acyl-CoA dehydrogenase)
1,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Linoleate monohydroperoxide (L-HPO), methyl linoleate monohydroperoxide (ML-HPO), and methyl hydroperoxy-epoxy-octadecenoate (ML-X) inhibited state 3 respiration of mitochondria when palmitate, palmitoyl CoA, or L-palmitoylcarnitine was used as a substrate. L-HPO was the most effective, and 50% inhibition of palmitate-supported respiration was observed with 2, 3.3, and 6.5 nmol/mg protein of L-HPO, ML-X, and ML-HPO, respectively. Almost the same values were obtained when palmitoyl CoA or L-palmitoylcarnitine was used in place of palmitate. L-HPO inhibited the reaction of beta-oxidation in mitochondria in a similar concentration range (4 nmol/mg protein for 50% inhibition) when L-palmitoylcarnitine was used as a substrate. L-HPO also inhibited the formation of 3-hydroxypalmitoylcarnitine from the same substrate. Carnitine palmitoyltransferase activity of mitochondria was inhibited by L-HPO, 50% inhibition occurring at 12 nmol/mg protein. These inhibitory effects of L-HPO were weaker when ATP was removed by hexokinase and glucose. ATP-dependent formation of carnitine ester of L-HPO was also suggested. It was deduced that L-HPO (and ML-X and ML-HPO after hydrolysis) was converted to carnitine ester and inhibited the palmitate metabolism at the site(s) of intramitochondrial carnitine palmitoyltransferase (and possibly acyl CoA dehydrogenase).
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PMID:Inhibition of palmitate oxidation in mitochondria by lipid hydroperoxides. 672 34

The toxicity of most drugs and chemicals is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of organs and organelles, including mitochondria. The toxicity of haloalkene-derived cysteine S-conjugates and related 4-thiaalkanoates is associated with their mitochondrial bioactivation. Toxic cysteine S-conjugates are formed by the glutathione S-transferase-catalyzed addition of glutathione to haloalkenes to give glutathione S-conjugates, which are hydrolyzed by gamma-glutamyltransferase and dipeptidases. Mitochondrial cysteine conjugate beta-lyase-catalyzed bioactivation of cysteine S-conjugates affords unstable alpha-halothiolates. Haloalkene-derived 4-thiaalkanoates, which are analogs of cysteine S-conjugates that lack an alpha-amino group, undergo bioactivation by the enzymes of fatty acid beta-oxidation to give 3-hydroxy-4-thiaalkanoates that eliminate alpha-halothiolates. alpha-Halothiolates yield alkylating and acylating agents that interact with cellular macromolecules and thereby cause cell damage. Mitochondrial dysfunction is the hallmark of cysteine S-conjugate-induced cytotoxicity: decreased respiration, decreased ATP and total adenine nucleotide concentrations, depletion of the mitochondrial glutathione content, perturbations in cellular Ca2+ homeostasis, and damage to the mitochondrial genome are seen with cysteine S-conjugates. Similar changes are observed with cytotoxic 4-thiaalkanoates, but inhibition of the medium-chain acyl-CoA dehydrogenase and hypoglycemia are also observed.
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PMID:Mitochondrial bioactivation of cysteine S-conjugates and 4-thiaalkanoates: implications for mitochondrial dysfunction and mitochondrial diseases. 759 25

We studied the role of FAD in the intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD), a homotetrameric mitochondrial enzyme containing a molecule of non-covalently bound FAD/monomer. In the MCAD molecule, FAD is buried in a crevice containing the active center. We have previously shown that upon import into mitochondria, newly processed MCAD is first incorporated into a high molecular weight (hMr) complex and that the hMr complex mainly consisted of MCAD-heat-shock protein 60 (hsp60) complex (Saijo, T., Welch, W.J., and Tanaka, K (1994) J. Biol. Chem. 269, 4401-4408). In the present study, we incubated in vitro synthesized precursor MCAD with mitochondria isolated from normal and riboflavin-deficient rat liver for 10-60 min and fractionated the solubilized mitochondria using gel filtration. The amount of MCAD in the hMr complex was larger and that of tetramer was smaller in riboflavin-deficient mitochondria than in control at any time point. In addition, riboflavin-deficient mitochondria were solubilized after 10-min import in a buffer containing ATP and were chased in the presence of FAD, FMN, or NAD+ or without any addition. The mitochondrial proteins were analyzed using gel filtration or immunoprecipitated with anti-hsp60 antibody. After 60-min chase in the presence of FAD, the majority of MCAD in the complex with hsp60 was transferred to tetramer, whereas no such transfer occurred after the chase in the absence of FAD. When chase was done in the presence of FMN, a significant amount of MCAD was transferred from the complex with hsp60 to tetramer, but the transfer was not as efficient as in the presence of FAD. The chase in the presence of NAD+ resulted in no transfer. These data suggest that isoalloxazine ring of FAD plays a critical role, exerting nucleating effect, in the hsp60-assisted folding of MCAD subunit into an assembly competent conformation, probably assisting the formation of the core.
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PMID:Isoalloxazine ring of FAD is required for the formation of the core in the Hsp60-assisted folding of medium chain acyl-CoA dehydrogenase subunit into the assembly competent conformation in mitochondria. 782 28

We incubated in vitro translated precursor of medium-chain acyl-CoA dehydrogenase (MCAD) with isolated rat liver mitochondria and fractionated the solubilized mitochondria on gel filtration. After a 5-min import into mitochondria, MCAD was recovered exclusively as a high molecular weight (hMr) complex (700,000), while after a 10-min import, it was recovered mainly in the hMr complex and mature tetramer, with a small amount in monomer. Either a further 15-min chase or exposure to ATP caused a marked decrease of MCAD in the hMr complex and an increase in the mature tetramer in comparable amounts, suggesting that the hMr complex was the precursor of tetramer. No monomer was detected in either case. Using specific antibodies, we have shown that the hMr complex represented a complex of MCAD and heat-shock protein 60 (hsp60), and, that upon import into mitochondria, unfolded MCAD first formed a transient complex with mitochondrial heat-shock protein 70 (hsp70mit) and then transferred to hsp60 to complete its folding into an assembly-competent conformation. We also examined the assembly of K304E MCAD, which is a prevalent variant enzyme among patients with MCAD deficiency. The assembly of the K304E into its tetrameric form was severely impaired. The binding of K304E with hsp70mit and its transfer from hsp70mit to hsp60 were normal. However, the hsp60 complex of K304E was much more stable than the wild-type counterpart upon a 15-min chase or exposure to ATP, suggesting that the folding in, or the transfer of K304E subunit to tetramer from, the complex with hsp60 was impaired.
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PMID:Intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD). Demonstration of impaired transfer of K304E-variant MCAD from its complex with hsp60 to the native tetramer. 790 78

Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.
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PMID:Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA. 824 Nov 27

Mammalian electron-transferring flavoprotein (ETF) has been reported to consist of two non-identical subunits and one FAD. The present paper shows that ETF purified from pig kidney contains one more molecule, an AMP. ETF was denatured by guanidine hydrochloride and ultrafiltered for the purpose of removing proteins. The filtrate was analyzed by reverse-phase chromatography. Two peaks appeared on the chromatogram: they were identified as FAD and AMP, and their molar amounts were identical, indicating that ETF contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contain one AMP molecule. These results clearly demonstrate that ETF has an AMP-binding site in addition to the FAD-binding site. AMP-free apoETF was prepared by guanidine treatment of ETF. Mixing AMP-free apoETF, FAD, and AMP produced reconstituted ETF, which showed the same properties as native ETF. Mixing AMP-free apoETF and FAD produced AMP-free ETF, regardless of the coexistence of ATP or ADP: the AMP-binding site cannot bind FAD, ADP, or ATP. The enzymatic activity of the AMP-free ETF for electron transfer from substrate-reduced medium-chain acyl-CoA dehydrogenase to 2,6-dichlorophenolindophenol was identical to that of native ETF. This indicates that the AMP contained in holoETF has no apparent influence on this enzymatic activity. A role of AMP recognized in this study is that AMP facilitates the formation of holoETF from AMP-free apoETF, FAD, and AMP.
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PMID:Electron-transferring flavoprotein has an AMP-binding site in addition to the FAD-binding site. 826 2

Skin fibroblast carnitine uptake studies may identify and differentiate primary and secondary carnitine deficiency disorders. To confirm the specificity of these studies in differentiating primary from secondary carnitine deficiency disorders, we have studied carnitine uptake in the cultured skin fibroblasts from 5 children who have various enzymatic defects in intramitochondrial beta-oxidation including short-chain, medium-chain and long-chain acyl-CoA dehydrogenase and short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiencies, and in 4 children with cytochrome oxidase deficiency. Carnitine uptake was normal in the intramitochondrial beta-oxidation cases, suggesting other mechanisms for their carnitine deficiency. Therefore, intramitochondrial beta-oxidation defects associated with carnitine deficiency can be differentiated from primary carnitine deficiency not only by the presence of an abnormal dicarboxylic aciduria but by normal skin fibroblast carnitine uptake. In contrast to these findings, carnitine uptake in the cultured skin fibroblasts of four children with secondary carnitine deficiency due to cytochrome oxidase deficiency demonstrated a partial decrease in the maximal velocity of uptake (20-47% control Vmax), similar to that observed in the primary carnitine deficiency heterozygotes. We propose that this observation may be due to a generalized decrease in intracellular ATP, thus decreasing the efficiency of the energy- and sodium-dependent carnitine transporter. We conclude that carnitine uptake studies in cultured skin fibroblasts will contribute to an understanding of the mechanisms of carnitine depletion in the primary and secondary carnitine deficiency disorders.
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PMID:Skin fibroblast carnitine uptake in secondary carnitine deficiency disorders. 838 12

Mitochondrial fatty acid beta-oxidation plays a major role in providing the ATP required for reabsorptive processes in the adult rat kidney. However, the molecular mechanisms and signals involved in induction of the enzymes of fatty acid oxidation during development in this and other organs are unknown. We therefore studied the changes in the steady-state levels of mRNA encoding medium-chain acyl-CoA dehydrogenase (MCAD), which catalyses the initial step in mitochondrial fatty acid beta-oxidation, in the rat kidney cortex and medulla between postnatal days 10 and 30. Furthermore, we investigated whether the expression of MCAD and of mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid cycle, might be co-ordinately regulated by circulating glucocorticoids in the immature kidney during development. In the cortex, the levels of MCAD mRNA rose 4-fold between day 10 and day 21, and then decreased from day 21 to day 30. In the medulla a postnatal increase in the concentration of MCAD mRNA (8-fold) was observed during the same period. Adrenalectomy prevented the 16-21-day developmental increases in MCAD and mMDH mRNA levels in the cortex and medulla; these could be restored by dexamethasone treatment. A single injection of dexamethasone into 10-day-old rats led to a rise in MCAD and mMDH mRNA levels in the renal cortex due to stimulation of gene transcription, as shown by nuclear run-on assays. Therefore MCAD and mMDH gene expression is tightly regulated at the transcriptional level by developmental changes in circulating glucocorticoid levels. These hormones might thus represent a good candidate as a co-ordinating factor in the expression of nuclear genes encoding mitochondrial enzymes in the kidney during postnatal development.
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PMID:Transcriptional regulation by glucocorticoids of mitochondrial oxidative enzyme genes in the developing rat kidney. 861 29

5-Hydroxydecanoate (5-HD) inhibits ischaemic and pharmacological preconditioning of the heart. Since 5-HD is thought to inhibit specifically the putative mitochondrial ATP-sensitive K+ (KATP) channel, this channel has been inferred to be a mediator of preconditioning. However, it has recently been shown that 5-HD is a substrate for acyl-CoA synthetase, the mitochondrial enzyme which 'activates' fatty acids. Here, we tested whether activated 5-HD, 5-hydroxydecanoyl-CoA (5-HD-CoA), is a substrate for medium-chain acyl-CoA dehydrogenase (MCAD), the committed step of the mitochondrial beta-oxidation pathway. Using a molecular model, we predicted that the hydroxyl group on the acyl tail of 5-HD-CoA would not sterically hinder the active site of MCAD. Indeed, we found that 5-HD-CoA was a substrate for purified human liver MCAD with a Km of 12.8 +/- 0.6 microM and a kcat of 14.1 s-1. For comparison, with decanoyl-CoA (Km approximately 3 microM) as substrate, kcat was 6.4 s-1. 5-HD-CoA was also a substrate for purified pig kidney MCAD. We next tested whether the reaction product, 5-hydroxydecenoyl-CoA (5-HD-enoyl-CoA), was a substrate for enoyl-CoA hydratase, the second enzyme of the beta-oxidation pathway. Similar to decenoyl-CoA, purified 5-HD-enoyl-CoA was also a substrate for the hydratase reaction. In conclusion, we have shown that 5-HD is metabolised at least as far as the third enzyme of the beta-oxidation pathway. Our results open the possibility that beta-oxidation of 5-HD or metabolic intermediates of 5-HD may be responsible for the inhibitory effects of 5-HD on preconditioning of the heart.
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PMID:Beta-oxidation of 5-hydroxydecanoate, a putative blocker of mitochondrial ATP-sensitive potassium channels. 1256 16

Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macro-molecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. Because of the high sensitivity achieved by the present proteomics technology, the purity of samples to be analyzed is important for the interpretation of the results obtained. In the present study, peroxisomes isolated from rat liver by usual cell fractionation were further purified by immunoisolation using a specific antibody raised against a peroxisomal membrane protein, PMP70. The isolated peroxisomes were analyzed by SDS-PAGE combined with liquid chromatography/mass spectrometry. Altogether 34 known peroxisomal proteins were identified in addition to several mitochondrial and microsomal proteins. Some of the latter may reside in the peroxisomes as well. Analysis of membrane fractions identified all known peroxins except for Pex7. Two new peroxisomal proteins of unknown function were of high abundance. One is a bi-functional protein consisting of an aminoglycoside phosphotransferase-domain and an acyl-CoA dehydrogenase domain. The other is a newly identified peroxisome-specific isoform of Lon protease, an ATP-dependent protease with chaperone-like activity. The peroxisomal localization of the protein was confirmed by immunological techniques. The peroxisome-type Lon protease, which is distinct from the mitochondrial isoform, may play an important role in the peroxisomal biogenesis.
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PMID:Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease. 1456 59


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