Procedure for enzymatic synthesis and isolation of radioactive long chain acyl-CoA esters

Banis, R. J.; Roberts, Cynthia; Stokes, Gilbert B.; Tove, S. B.

doi:10.1016/0003-2697(76)90135-4

Cited by 24 publications

(9 citation statements)

References 4 publications

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“…ACS specific activity was assayed in a 200 μL mixture containing 0.1 μCi [ 14 C]-palmitate (0,1 μCi/ μL; Perkin-Elmer,Waltham, USA), supplemented with 50 μM palmitate in 0.5 mM Triton X-100, 200 mM Tris (pH 7.4), 12 mM MgCl 2 , 1 mM EDTA, 5 mM dithiothreitol (DTT), 5 mM ATP, 250 mM CoA and 30 μg of micelles composed by phosphatidylcholine and phosphatidylserine (2:1; w/w) [31, 32]. The reaction was initiated by adding 0.1–6 μg of protein to the assay mixture.…”

Section: Methodsmentioning

confidence: 99%

Long-chain acyl-CoA synthetase 2 knockdown leads to decreased fatty acid oxidation in fat body and reduced reproductive capacity in the insect Rhodnius prolixus

Auger

Klett

Paula

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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Long-chain acyl-CoA esters are important intermediates in lipid metabolism and are synthesized from fatty acids by long-chain acyl-CoA synthetases (ACSL). The hematophagous insect Rhodnius prolixus, a vector of Chagas' disease, produces glycerolipids in the midgut after a blood meal, which are stored as triacylglycerol in the fat body and eggs. We identified twenty acyl-CoA synthetase genes in R. prolixus, two encoding ACSL isoforms (RhoprAcsl1 and RhoprAcsl2). RhoprAcsl1 transcripts increased in posterior midgut on the second day after feeding, and RhoprAcsl2 was highly transcribed on the tenth day. Both enzymes were expressed in Escherichia coli. Recombinant RhoprACSL1 and RhoprACSL2 had broad pH optima (7.5-9.5 and 6.5-9.5, respectively), were inhibited by triacsin C, and were rosiglitazone-insensitive. Both showed similar apparent Km for palmitic and oleic acid (2-6 μM), but different Km for arachidonic acid (0.5 and 6 μM for RhoprACSL1-Flag and RhoprACSL2-Flag, respectively). The knockdown of RhoprAcsl1 did not result in noticeable phenotypes. However, RhoprACSL2 deficient insects exhibited a 2.5-fold increase in triacylglycerol content in the fat body, and 90% decrease in fatty acid β-oxidation. RhoprAcsl2 knockdown also resulted in 20% increase in lifespan, delayed digestion, 30% reduced oviposition, and 50% reduction in egg hatching. Laid eggs and hatched nymphs showed remarkable alterations in morphology. In summary, R. prolixus ACSL isoforms have distinct roles on lipid metabolism. Although RhoprACSL1 functions remain unclear, we propose that RhoprACSL2 is the main contributor for the formation of the intracellular acyl-CoA pool channeled for β-oxidation in the fat body, and is also required for normal reproduction.

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Section: Methodsmentioning

confidence: 99%

Long-chain acyl-CoA synthetase 2 knockdown leads to decreased fatty acid oxidation in fat body and reduced reproductive capacity in the insect Rhodnius prolixus

Auger

Klett

Paula

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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“…14 C]palmitic acid, 10 mM ATP, 5 mM dithiothreitol, and 0.25 mM CoA for 10 min at 37°C (17). DGAT activity was assayed using 5-20 g of protein, 0.2 mM sn-1,2-dioleoylglycerol, and 30 M [ 3 H]palmitoyl-CoA for 10 min at room temperature (18).…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Glycerol Phosphate Acyltransferase Directs the Incorporation of Exogenous Fatty Acids into Triacylglycerol

Igal

Wang

González-Baró

et al. 2001

Journal of Biological Chemistry

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The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [ 14 C]oleate (ϳ3 M), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [ 14 C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 M oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.Excess accumulation of triacylglycerol in adipocytes is a major factor contributing to the current epidemic of obesity-related disorders, including type 2 diabetes, and excess accumulation of triacylglycerol in non-adipocytes has been linked to lipid-mediated apoptosis (1, 2). Although obesity research has focused on the storage of calories that are ingested in excess of energy needs, the observation that mice null for diacylglycerol acyltransferase are resistant to diet-induced obesity suggests the possibility that, independent of energy intake or expenditure, the amount of triacylglycerol stored can be regulated biochemically (3). This remarkable observation further suggests that the synthesis of triacylglycerol can be specifically regulated and that regulation of the synthetic p...

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“…Enzymatic acylations of CoA (eq. 7) using AcCoA synthetase (EC 6.2.1.1) or fatty acyl CoA synthetase (fatty acid: CoA ligase, EC 6.2.1.3) are highly selective for S-acylation but have been demonstrated in preparations of only 1-25 !mot (Kornberg and Pricer, 1953a;Gaillard and Stumpfe, 1968;Branis et al, 1976;Merrill, Gidwitz and Bell, 1982). The substrate specificity of both enzymes is broad (Tanaka etal., 1979;Patel and Walt, 1987).…”

Section: Preparation Of Acyl-coamentioning

confidence: 95%

Cofactor Regeneration for Enzyme-Catalysed Synthesis

Chenault

Simon

Whitesides

1988

Biotechnology and Genetic Engineering Reviews

200

126

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Procedure for enzymatic synthesis and isolation of radioactive long chain acyl-CoA esters

Cited by 24 publications

References 4 publications

Long-chain acyl-CoA synthetase 2 knockdown leads to decreased fatty acid oxidation in fat body and reduced reproductive capacity in the insect Rhodnius prolixus

Long-chain acyl-CoA synthetase 2 knockdown leads to decreased fatty acid oxidation in fat body and reduced reproductive capacity in the insect Rhodnius prolixus

Mitochondrial Glycerol Phosphate Acyltransferase Directs the Incorporation of Exogenous Fatty Acids into Triacylglycerol

Cofactor Regeneration for Enzyme-Catalysed Synthesis

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