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1.
We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [13C2,2H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [13C6]glucose or [1,2-13C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-13C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.  相似文献   

2.
Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-14C]malonyl-CoA was incorporated into fatty acids of C16C26. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C12C24) elongation showed that stearoyl-CoA, in the presence of [2-14C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C20C26. [1-14C]C16CoA and [1-14C]C18CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-14C]C18CoA was a better primer than [1-14C]C16CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-14C]C18CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-14C]malonyl-CoA in the presence of C18CoA, on the one hand, and [1-14C]C18CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.  相似文献   

3.
Rat hearts perfused for up to 60 min in the working mode with palmitate, but not with glucose, resulted in substantial formation of palmitoylcarnitine and stearoylcarnitine. To test whether lipolysis of endogenous lipids was responsible for the increased stearoylcarnitine content or whether some of the perfused palmitate underwent chain elongation, hearts were perfused with hexadecanoic-16,16,16-d(3) acid (M+3). The pentafluorophenacyl ester of deuterium labeled stearoylcarnitine had an M+3 (639.4 m/z) compared to the unlabeled M+0 (636.3 m/z) consistent with a direct chain elongation of the perfused palmitate. Furthermore, the near equal isotope enrichment of palmitoyl- (90.2+/-5.8%) and stearoylcarnitine (78.0+/-7.1%) suggest that both palmitoyl- and stearoyl-CoA have ready access to mitochondrial carnitine palmitoyltransferase and that most of the stearoylcarnitine is derived from the perfused palmitate.  相似文献   

4.
PhlD, a type III polyketide synthase from Pseudomonas fluorescens, catalyzes the synthesis of phloroglucinol from three molecules of malonyl-CoA. Kinetic analysis by direct measurement of the appearance of the CoASH product (k(cat) = 24 +/- 4 min(-1) and Km = 13 +/- 1 microM) gave a k(cat) value more than an order of magnitude higher than that of any other known type III polyketide synthase. PhlD exhibits broad substrate specificity, accepting C4-C12 aliphatic acyl-CoAs and phenylacetyl-CoA as the starters to form C6-polyoxoalkylated alpha-pyrones from sequential condensation with malonyl-CoA. Interestingly, when primed with long chain acyl-CoAs, PhlD catalyzed extra polyketide elongation to form up to heptaketide products. A homology structural model of PhlD showed the presence of a buried tunnel extending out from the active site to assist the binding of long chain acyl-CoAs. To probe the structural basis for the unusual ability of PhlD to accept long chain acyl-CoAs, both site-directed mutagenesis and saturation mutagenesis were carried out on key residues lining the tunnel. Three mutations, M21I, H24V, and L59M, were found to significantly reduce the reactivity of PhlD with lauroyl-CoA while still retaining its physiological activity to synthesize phloroglucinol. Our homology modeling and mutational studies indicated that even subtle changes in the tunnel volume could affect the ability of PhlD to accept long chain acyl-CoAs. This suggested novel strategies for combinatorial biosynthesis of unnatural pharmaceutically important polyketides.  相似文献   

5.
Peroxisomes play an essential role in a number of important metabolic pathways including β-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various β-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal β-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp249 to Arg251) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp249 to Arg251 and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.  相似文献   

6.
The shuttle system that mediates the transport of fatty acids across the mitochondrial membrane in invertebrates has received little attention. Carnitine O-palmitoyltransferase I (EC 2.3.1.21; CPT I) is a key component of this system that in vertebrates controls long-chain fatty acid β-oxidation. To gain knowledge on the acyltransferases in aquatic arthropods, physical, kinetic, regulatory and immunological properties of CPT of the midgut gland mitochondria of Macrobrachium borellii were assayed. CPT I optimum conditions were 34 °C and pH = 8.0. Kinetic analysis revealed a Km for carnitine of 2180 ± 281 μM and a Km for palmitoyl-CoA of 98.9 ± 8.9 μM, while Vmax were 56.5 ± 6.6 and 36.7 ± 4.8 nmol min− 1 mg protein− 1, respectively. A Hill coefficient, n ~ 1, indicate a Michaelis–Menten behavior. The CPT I activity was sensitive to regulation by malonyl-CoA, with an IC50 of 25.2 μM. Electrophoretic and immunological analyses showed that a 66 kDa protein with an isoelectric point of 5.1 cross-reacted with both rat liver and muscle-liver anti CPT I polyclonal antibodies, suggesting antigenic similarity with the rat enzymes. Although CPT I displayed kinetic differences with insect and vertebrates, prawn showed a high capacity for energy generation through β-oxidation of long-chain fatty acids.  相似文献   

7.
Little is known about the sources of acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of mitochondrial fatty acid oxidation in the heart. In perfused rat hearts, we previously showed that malonyl-CoA is labeled from both carbohydrates and fatty acids. This study was aimed at assessing the mechanisms of incorporation of fatty acid carbons into malonyl-CoA. Rat hearts were perfused with glucose, lactate, pyruvate, and a fatty acid (palmitate, oleate or docosanoate). In each experiment, substrates were (13)C-labeled to yield singly or/and doubly labeled acetyl-CoA. The mass isotopomer distribution of malonyl-CoA was compared with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of labeled glucose or lactate/pyruvate, the (13)C labeling of malonyl-CoA was up to 2-fold lower than that of mitochondrial acetyl-CoA. However, in the presence of a fatty acid labeled in its first acetyl moiety, the (13)C labeling of malonyl-CoA was up to 10-fold higher than that of mitochondrial acetyl-CoA. The labeling of malonyl-CoA and of the acetyl moiety of citrate is compatible with peroxisomal beta-oxidation forming C(12) and C(14) acyl-CoAs and contributing >50% of the fatty acid-derived acetyl groups that end up in malonyl-CoA. This fraction increases with the fatty acid chain length. By supplying acetyl-CoA for malonyl-CoA synthesis, peroxisomal beta-oxidation may participate in the control of mitochondrial fatty acid oxidation in the heart. In addition, this pathway may supply some acyl groups used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes.  相似文献   

8.
During the glyoxysomal β-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125–130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal β-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the β-oxidation of acyl-CoAs of various chain lengths showed that the β-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 μM. At substrate concentrations >20 μM, long-chain acyl-CoAs were β-oxidized more slowly than short-chain acyl-CoAs because the β-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5–10 μM substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the β-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the β-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (≤5 μM) of a long-chain acyl-CoA exerted an inhibitory effect on the β-oxidation rate of butyryl-CoA. Reversibility of the inhibition was observed as well as metabolization of the inhibiting long-chain acyl-CoA. Regarding the activities of the individual β-oxidation enzymes towards their C4 substrates in the presence of a long-chain acyl-CoA, the MFP activity exhibited strong inhibition. This inhibition appears not to be due to the detergent-like physical properties of long-chain acyl-CoAs. The results of the studies, which are consistent with the observation that short-chain intermediates accumulate transiently during complete degradation of a long-chain acyl-CoA, suggest that the substrate concentration-dependent chain-length specificity of the β-oxidation and a metabolic regulation at the level of MFP are factors determining this transient accumulation. Received: 2 February 1999 / Accepted: 14 April 1999  相似文献   

9.
A soluble enzyme system from the stroma of chloroplasts isolated from Spinacease oleraceae elongated various long chain acyl-CoAs using acetyl-CoA as a two-carbon donor. Partial purification of the system was achieved by ammonium sulfate fractionation and molecular sieve chromatography. The elongation system required NADPH and NADH for the reduction steps. Several nucleoside triphosphates markedly stimulated elongation. Inhibition occurred with several thiol binding reagents and with free CoA. The possible significance of elongation via acyl-CoAs in chloroplasts is discussed.  相似文献   

10.
The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.  相似文献   

11.
[1-14C]acetate and [2-14C]acetate were incorporated into the β-diketones of barley spike epicuticular wax via the peduncle. Utilizing column chromatography with dry copper acetate, the β-diketones were isolated and the labeling pattern in the hentriacontan-14, 16-dione determined after its degradation. A modified iodoform procedure was used to give myristic and palmitic acids. Radio-gas chromatography was then performed on the products of chemical α-oxidation of the separated fatty acids. This procedure, in effect, gave the specific activity of every carbon atom of hentriacontan-14,16-dione except carbon-1 to carbon-5 (from myristic acid) and carbon-27 to carbon-31 (from palmitic acid) for each labeled substrate. The specific activity of carbon-15 was determined by an indirect method. On the basis of these data it is suggested that the hentriacontan-14,16-dione is synthesized from the carbon-31 end of the molecule by elongation as follows. C2 units are added, perhaps to a mixture of short chain precursors, to give a chain with 12 carbon atoms. This chain is then elongated to one with 16 carbon atoms so that the four added carbon atoms are uniformly labeled. Following this, the chain with 16 carbon atoms is elongated with C2 units to give the complete molecule. Possibly some change in mechanism occurs in this last elongation process when the chain is 22 carbon atoms long. Barley spike wax β-diketones contain about 2% nonacosan-13, 15-dione which seems to be synthesized in an analogous manner.  相似文献   

12.
Very long-chain fatty acids (VLCFAs, chain length >C20) exist in tissues throughout the body and are synthesized by repetition of the fatty acid (FA) elongation cycle composed of four successive enzymatic reactions. In mammals, the TER gene is the only gene encoding trans-2-enoyl-CoA reductase, which catalyzes the fourth reaction in the FA elongation cycle. The TER P182L mutation is the pathogenic mutation for nonsyndromic mental retardation. This mutation substitutes a leucine for a proline residue at amino acid 182 in the TER enzyme. Currently, the mechanism by which the TER P182L mutation causes nonsyndromic mental retardation is unknown. To understand the effect of this mutation on the TER enzyme and VLCFA synthesis, we have biochemically characterized the TER P182L mutant enzyme using yeast and mammalian cells transfected with the TER P182L mutant gene and analyzed the FA elongation cycle in the B-lymphoblastoid cell line with the homozygous TER P182L mutation (TERP182L/P182L B-lymphoblastoid cell line). We have found that TER P182L mutant enzyme exhibits reduced trans-2-enoyl-CoA reductase activity and protein stability, thereby impairing VLCFA synthesis and, in turn, altering the sphingolipid profile (i.e. decreased level of C24 sphingomyelin and C24 ceramide) in the TERP182L/P182L B-lymphoblastoid cell line. We have also found that in addition to the TER enzyme-catalyzed fourth reaction, the third reaction in the FA elongation cycle is affected by the TER P182L mutation. These findings provide new insight into the biochemical defects associated with this genetic mutation.  相似文献   

13.
Effects of dietary conjugated linoleic acid (CLA, 1% mixed isomers) on n-6 long-chain polyunsaturated fatty acid (LCPUFA) oxidation and biosynthesis were investigated in liver and brain tissues of neonatal piglets. Fatty acid β-oxidation was measured in tissue homogenates using [1-14C]linoleic acid (LA) and -arachidonic acid (ARA) substrates, while fatty acid desaturation and elongation were traced using [U-13C]LA and GC-MS. Dietary CLA had no effect on fatty acid β-oxidation, but significantly decreased n-6 LCPUFA biosynthesis by inhibition of LA elongation and desaturation. Differences were noted between our 13C tracer assessment of desaturation/elongation and simple precursor-product indices computed from fatty acid composition data, indicating that caution should be exercised when employing the later. The inhibitory effects of CLA on elongation/desaturation were more pronounced in pigs fed a low fat diet (3% fat) than a high fat diet (25% fat). Direct elongation of linoleic acid to C20:2n-6 via the alternate elongation pathway might play an important role in n-6 LCPUFA synthesis because more than 40% of the synthetic products of [U-13C]LA accumulated in [13C]20:2n-6. Overall, the data show that dietary CLA shifted the distribution of the synthetic products of [U-13C]LA between elongation and desaturation in liver and decreased the total synthetic products of [U-13C]LA in brain by inhibiting LA elongation to C20:2n-6. The impact of CLA on brain LCPUFA metabolism of the developing neonate merits consideration and further investigation.  相似文献   

14.
This study describes the use of a stable-isotope labeled precursor ([U-13C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (13C appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-13C]palmitic acid, ~60% of the total palmitoyl-CoA was 13C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelins were 62 ± 3, 13 ± 2, and 60 ± 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 ± 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ~1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations.  相似文献   

15.
Complex I is responsible for most of the mitochondrial H2O2 release, low during the oxidation of the NAD linked substrates and high during succinate oxidation, via reverse electron flow. This H2O2 production appear physiological since it occurs at submillimolar concentrations of succinate also in the presence of NAD substrates in heart (present work) and rat brain mitochondria (Zoccarato et al., Biochem J, 406:125–129, 2007). Long chain fatty acyl-CoAs, but not fatty acids, act as strong inhibitors of succinate dependent H2O2 release. The inhibitory effect of acyl-CoAs is independent of their oxidation, being relieved by carnitine and unaffected or potentiated by malonyl-CoA. The inhibition appears to depend on the unbound form since the acyl-CoA effect decreases at BSA concentrations higher than 2 mg/ml; it is not dependent on ΔpH or Δp and could depend on the inhibition of reverse electron transfer at complex I, since palmitoyl-CoA inhibits the succinate dependent NAD(P) or acetoacetate reduction.  相似文献   

16.
Straight chain fatty acid α-oxidation increases during differentiation of 3T3-L1 adipocytes, leading to a marked accumulation of odd chain length fatty acyl moieties. Potential roles of this pathway in adipocyte differentiation and lipogenesis are unknown. Mammalian fatty acid 2-hydroxylase (FA2H) was recently identified and suggested to catalyze the initial step of straight chain fatty acid α-oxidation. Accordingly, we examined whether FA2H modulates adipocyte differentiation and lipogenesis in mature adipocytes. FA2H level markedly increases during differentiation of 3T3-L1 adipocytes, and small interfering RNAs against FA2H inhibit the differentiation process. In mature adipocytes, depletion of FA2H inhibits basal and insulin-stimulated glucose uptake and lipogenesis, which are partially rescued by the enzymatic product of FA2H, 2-hydroxy palmitic acid. Expression of fatty-acid synthase and SCD1 was decreased in FA2H-depleted cells, and levels of GLUT4 and insulin receptor proteins were reduced. 2-Hydroxy fatty acids are enriched in cellular sphingolipids, which are components of membrane rafts. Accelerated diffusional mobility of raft-associated lipids was shown to enhance degradation of GLUT4 and insulin receptor in adipocytes. Consistent with this, depletion of FA2H appeared to increase raft lipid mobility as it significantly accelerated the rates of fluorescence recovery after photobleaching measurements of lipid rafts labeled with Alexa 488-conjugated cholera toxin subunit B. Moreover, the enhanced recovery rates were partially reversed by treatment with 2-hydroxy palmitic acid. In conclusion, our findings document the novel role of FA2H in adipocyte lipogenesis possibly by modulation of raft fluidity and level of GLUT4.  相似文献   

17.
We investigated the compartmentation of the catabolism of dodecanedioate (DODA), azelate, and glutarate in perfused rat livers, using a combination of metabolomics and mass isotopomer analyses. Livers were perfused with recirculating or nonrecirculating buffer containing one fully 13C-labeled dicarboxylate. Information on the peroxisomal versus mitochondrial catabolism was gathered from the labeling patterns of acetyl-CoA proxies, i.e. total acetyl-CoA, the acetyl moiety of citrate, C-1 + 2 of β-hydroxybutyrate, malonyl-CoA, and acetylcarnitine. Additional information was obtained from the labeling patterns of citric acid cycle intermediates and related compounds. The data characterize the partial oxidation of DODA and azelate in peroxisomes, with terminal oxidation in mitochondria. We did not find evidence of peroxisomal oxidation of glutarate. Unexpectedly, DODA contributes a substantial fraction to anaplerosis of the citric acid cycle. This opens the possibility to use water-soluble DODA in nutritional or pharmacological anaplerotic therapy when other anaplerotic substrates are impractical or contraindicated, e.g. in propionic acidemia and methylmalonic acidemia.  相似文献   

18.
The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (Mr 70,000) and FATP2b (Mr 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.  相似文献   

19.
The elongation of [9,10-3H]oleoyl-CoA with malonyl-CoA to form 20, 22, and 24 carbon monounsaturated fatty acids was demonstrated in housefly microsomes by radio-GLC. These elongation reactions, which have been postulated to be involved in hydrocarbon biosynthesis, have not been previously demonstrated in insects. 2-Octadecynoate (18:1 Δ2=) inhibited the in vivo incorporation of [1-14C]acetate into both fatty acids and hydrocarbons in a dose-dependent manner. At doses of 10 μg per female housefly of the alkynoic acid, the incorporation of [1-14C]acetate into hydrocarbon was inhibited 93%, the incorporation of [9,10-3H]oleate into hydrocarbon was inhibited 64%, and the incorporation of [1-14C]acetate into total internal lipid was inhibited 65%. Partially purified FAS was inhibited 50% and 95% at 15 μM and 40 μM, respectively, of the alkynoic acid. These results show that 2-octadecynoate inhibits hydrocarbon biosynthesis in the housefly by inhibiting FAS, and the in vivo data suggest that the elongation of 18:1 to longer chain fatty acids is also inhibited.  相似文献   

20.
In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-14C]18:0)-CoA was synthesized from [1-14C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-14C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-14C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [14C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.  相似文献   

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