Measurement of the acyl-CoA intermediates of <i>β</i>-oxidation by h.p.l.c. with on-line radiochemical and photodiode-array detection. Application to the study of [U-14C]hexadecanoate oxidation by intact rat liver mitochondria

Watmough, Nicholas J.; Turnbull, D. M.; Sherratt, H. S. A.; Bartlett, K.

doi:10.1042/bj2620261

Cited by 63 publications

(43 citation statements)

References 57 publications

(32 reference statements)

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“…Since mutations in ELOVL4 occur downstream of the active site histidines, the possibility remains that the truncated gene product may be enzymatically active, generating keto intermediates of fatty acid elongation in cellular compartments where it may be toxic. Although the hydroxyl and the enoyl acyl-CoA intermediates are amenable to derivatization and detection by HPLC, detection of the keto intermediate is problematic due to the lack of an available hydroxyl or carboxylic group for derivatization and organic acid analysis (44). The only methylketone ever detected is a 2-butanone resulting from β-ketothiolase enzyme deficiency (45).…”

Section: Discussionmentioning

confidence: 99%

Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy

Logan¹,

Agbaga

Chan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Autosomal-dominant Stargardt-like macular dystrophy [Stargardt3 (STGD3)] results from single allelic mutations in the elongation of very-long-chain fatty acids-like 4 (ELOVL4), whereas recessive mutations lead to skin and brain dysfunction. ELOVL4 protein localizes to the endoplasmic reticulum, where it mediates the condensation reaction catalyzing the formation of very-long-chain (VLC) (C-28 to C-40) fatty acids, saturated and polyunsaturated (PUFA). The defective gene product is truncated at the C terminus, leading to mislocalization and aggregation in other organelles. We hypothesized that the STGD3 truncated mutant may generate mislocalized, and therefore toxic, keto intermediates of fatty acid elongation, thereby contributing to the disease process. Using cell-based and cell-free microsome assays, we found that the truncated protein lacked innate condensation activity. Coexpression of different forms of wild-type and mutant ELOVL4 revealed a large dominant-negative effect of mutant protein on ELOVL4 localization and enzymatic activity, resulting in reduced VLC-PUFA synthesis. The reduction in VLC-PUFA levels in STGD3 and age-related macular degeneration may be a contributing factor to their retinal pathology.Stargardt disease | condensing enzyme | photoreceptor degeneration

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Section: Discussionmentioning

confidence: 99%

Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy

Logan¹,

Agbaga

Chan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…In contrast to perchloric acid, glacial acetic acid does not cause protein precipitation and allows a more easy transfer of the reaction mixture to an extraction tube. For extraction and subsequent HPLC analysis (modified from Watmough et al [30]) of the fatty acids and acyl-CoA derivatives, SO pl 0.25 mM [9,10-3H]palmitoyl-CoA (acyl-CoA internal standard, specific radioactivity 0.7 Ci/mmol) and SO p1 0.1 mM [9,10-'H]stearate (fatty acid internal standard, specific radioactivity 2.8 Cilmmol) were added to the reaction mixture, which was then transferred from the reaction vial to an extraction tube containing 250 p1 cold 12% (mass/vol.) HCIO,.…”

Section: -'"C]heptadecanoic Acid [I I ]mentioning

confidence: 99%

α‐Oxidation of 3‐Methyl‐Substituted Fatty Acids in Rat Liver

Croes

Casteels

Hoffmann

et al. 1996

European Journal of Biochemistry

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coxidation of 3-methyl-substituted fatty acids in rat liver was studied in intact and permeabilized rat hepatocytes, and in homogenates and subcellular fractions. The experiments revealed that the primary end product of a-oxidation is formic acid, which is then converted to CO,. Rates of a-oxidation identical to those observed in intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fez+, 2-oxoglutarate and ascorbate were added, suggesting that aoxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation by differential and density gradient centrifugation demonstrated that a-oxidation is confined to peroxisomes, which produce formic acid that is converted to CO,, mainly in the cytosol. a-Oxidation in broken cell systems went hand in hand with the formation of a 2-hydroxy-3-methylacyl-CoA ester. Formation of the metabolite was strictly dependent on the presence of the above-mentioned cofactors, was confined to peroxisomes and was inhibited by fenoprofen and propyl gallate, inhibitors of a-oxidation in intact cells, indicating that the 2-hydroxyacyl-CoA ester is a bona fide intermediate of a-oxidation. Selective omission of cofactors from the reaction mixture and analysis of the incubation mixtures for 3-methyl fatty acids, 3-methyl fatty acyl-CoAs and their respective 2-hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction.Our experiments demonstrate that a-oxidation is a peroxisomal process that consists of at least three reactions : fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid.

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“…Stearic acid, as well as stearoyl-CoA, were were incubated with mitochondrial outer membranes (4.8 ,ug protein) as described (see section 2.4.1.). Acyl carnitine was separated from acyl-CoA by DEAE-Sephacyl column chromatography [22], and radiolabelled acyl carnitine produced from radiolabelled fatty acid or fatty acyl-CoA was determined. The values, in nmol/min/mg protein, represent an average of two determinations.…”

Section: Resultsmentioning

confidence: 99%

“…In incubations where radiolabelled fatty acid was used as a substrate, the unreacted substrate was removed by extraction with hexane. Radiolabelled fatty acyl-CoA was separated from fatty acyl carnitine by DEAE-Sephacyl column (6 x 80 mm) chromatography [22]. Alternatively, after evaporation of the solvent, the reaction product was chromatographed on TLC silica gel 60 plates with butanol/water/acetic acid (60: 20: 7, by volume) as the solvent.…”

Section: Carnitine Acyl Tran~ferasementioning

confidence: 99%

Substrate specificity of rat liver mitochondrial carnitine palmitoyl transferase I: evidence against α‐oxidation of phytanic acid in rat liver mitochondria

Singh

Poulos

1995

FEBS Letters

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The two branched chain fatty acids pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) were converted to coenzyme A thioesters by rat liver mitochondrial outer membranes. However, these branched chain fatty acids could not be converted to pristanoyl and phytanoyl carnitines, respectively, by mitochondrial outer membranes. As expected, the unbranched long chain fatty acids, stearic acid and palmitic acid, were rapidly converted to stearoyl and palmitoyl carnitines, respectively, by mitochondrial outer membranes. These observations indicate that the branched chain fatty acids could not be transported into mitochondria. The data presented strongly suggest that in rat liver, c~-oxidation of phytanic acid occurs in organelles other than mitochondria.demonstrated that CPTI activity is reversibly inhibited by malonyl-CoA. Recently, it has been shown that malonyl-CoA inhibits peroxisomal, microsomal and plasma membrane carnitine acyl transferases [8,10,13]. Therefore, in order to understand the role of CPTI in fatty acid oxidation, CPTI must be separated from other carnitine acyl transferases. In view of the difficulties in isolating CPTI in the active state, we decided to investigate CPTI activity in highly purified mitochondrial outer membranes from rat liver. We present evidence that branched chain fatty acids with ~-or fl-methyl groups are poor substrates for CPTI, suggesting that these branched chain fatty acids cannot be transported into mitochondria via the carnitine/ acylcarnitine transport system.

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Measurement of the acyl-CoA intermediates of β-oxidation by h.p.l.c. with on-line radiochemical and photodiode-array detection. Application to the study of [U-14C]hexadecanoate oxidation by intact rat liver mitochondria

Cited by 63 publications

References 57 publications

Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy

Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy

α‐Oxidation of 3‐Methyl‐Substituted Fatty Acids in Rat Liver

Substrate specificity of rat liver mitochondrial carnitine palmitoyl transferase I: evidence against α‐oxidation of phytanic acid in rat liver mitochondria

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