A simple enzymatic method for the preparation of radiolabeled erucoyl-CoA and other long-chain fatty acyl-CoAs and their characterization by mass spectrometry

Taylor, David C.; Weber, Nikolaus; Hogge, Lawrence; Underhill, E. W.

doi:10.1016/0003-2697(90)90686-4

Cited by 106 publications

(70 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[9,10-3 H]Oleic acid (8 Ci mmol Ϫ1 ) was purchased from Amersham Pharmacia Biotech (Quebec). Labeled fatty acids were converted to the corresponding acyl-CoA thioesters using the method described by Taylor et al (1990). Specific activities were adjusted as required by diluting with authentic unlabeled standards.…”

Section: Substrates and Reagentsmentioning

confidence: 99%

Seed-Specific Over-Expression of an Arabidopsis cDNA Encoding a Diacylglycerol Acyltransferase Enhances Seed Oil Content and Seed Weight

et al. 2001

Self Cite

View full text Add to dashboard Cite

We recently reported the cloning and characterization of an Arabidopsis (ecotype Columbia) diacylglycerol acyltransferase cDNA (Zou et al., 1999) and found that in Arabidopsis mutant line AS11, an ethyl methanesulfonate-induced mutation at a locus on chromosome II designated as Tag1 consists of a 147-bp insertion in the DNA, which results in a repeat of the 81-bp exon 2 in the Tag1 cDNA. This insertion mutation is correlated with an altered seed fatty acid composition, reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol content, and delayed seed development in the AS11 mutant. The effect of the insertion mutation on microsomal acyl-coenzyme A-dependent DGAT is examined with respect to DGAT activity and its substrate specificity in the AS11 mutant relative to wild type. We demonstrate that transformation of mutant AS11 with a single copy of the wild-type Tag1 DGAT cDNA can complement the fatty acid and reduced oil phenotype of mutant AS11. More importantly, we show for the first time that seed-specific over-expression of the DGAT cDNA in wild-type Arabidopsis enhances oil deposition and average seed weight, which are correlated with DGAT transcript levels. The DGAT activity in developing seed of transgenic lines was enhanced by 10% to 70%. Thus, the current study confirms the important role of DGAT in regulating the quantity of seed triacylglycerols and the sink size in developing seeds.

show abstract

Section: Substrates and Reagentsmentioning

confidence: 99%

Seed-Specific Over-Expression of an Arabidopsis cDNA Encoding a Diacylglycerol Acyltransferase Enhances Seed Oil Content and Seed Weight

et al. 2001

Self Cite

View full text Add to dashboard Cite

show abstract

“…mtGPAT1 activity is calculated as the activity that is uninhibited by NEM. (45). ACAT specific activity was assayed in a 200-l mixture containing 50 g of total membrane protein, medium I, and 1.67 g/ml BSA, as described previously (42).…”

Section: Enzyme Assays Sn-[2-mentioning

confidence: 99%

Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes

Lewin¹,

Wang²,

Nagle³

et al. 2005

American Journal of Physiology-Endocrinology and Metabolism

View full text Add to dashboard Cite

(TAG) in nonadipose tissues is closely associated with the development of insulin resistance, interest has increased in the metabolism of longchain acyl-CoAs toward ␤-oxidation or the synthesis and storage of TAG. To learn whether a mitochondrial isoform of glycerol-3-phosphate acyltransferase (mtGPAT1) competes with carnitine palmitoyltransferase I (CPT I) for acyl-CoAs and whether it contributes to the formation of TAG, we overexpressed rat mtGPAT1 13-fold in primary hepatocytes obtained from fasted rats. When 100, 250, or 750 M oleate was present, both TAG mass and the incorporation of [ 14 C]oleate into TAG increased more than twofold in hepatocytes overexpressing mtGPAT1 compared with vector controls. Although the incorporation of [ 14 C]oleate into CO2 and acid-soluble metabolites increased with increasing amounts of oleate in the media, these metabolites were ϳ40% lower in the Ad-mtGPAT1 infected cells, consistent with competition for acyl-CoAs between CPT I and mtG-PAT1. A 50 -60% decrease was also observed in [14 C]oleate incorporation into cholesteryl ester. With increasing amounts of exogenous oleate, [14 C]TAG secretion increased appropriately in vector controlinfected hepatocytes, suggesting that the machinery for VLDL-TAG biogenesis and secretion was unaffected. Despite the marked increases in TAG synthesis and storage in the Ad-mtGPAT1 cells, however, the Ad-mtGPAT1 cells secreted the same amount of [ 14 C

show abstract

“…[l-14ClEicosenoic acid was synthesized as described previously . l-'4C-labeled fatty acids were converted to the corresponding acyl-COA thioesters using the method described by Taylor et al (1990 Mixed TAG and DAG standards for GC, which were not commercially available, were synthesized from the corresponding DAGs or MAGs by condensation with the appropriate acyl chloride and purified as described by . HPLC-grade solvents (Omni-Solv, BDH Chemicals, Toronto, ON) were used throughout these studies.…”

Section: Substrates and Reagentsmentioning

confidence: 99%

Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate-Induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity

et al. 1995

View full text Add to dashboard Cite

In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) i n the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS1 1, which, in comparison to wild type (WT), has reduced levels of 20:l and 18:l and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Cenetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAC1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity t o elongate 18:l or to an increase in the relative A1 5 or A1 2 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS1 1 seed homogenates. Rather, the fatty acid phenotype of AS1 1 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:l biosynthesis during seed development, leaving more 18:l available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result i n dramatic changes i n fatty acid composition and, in particular, i n the accumulation of VLCFAs i n seed storage lipids.The fatty acyl composition of seed TAGs determines their physical and chemical properties and, thus, their use in edible oil or industrial applications. TAG composition depends on the interaction of several different groups of enzymes in the lipid biosynthesis pathway. The enzymes of the fatty acid synthase complex in the plastids of developing seeds are responsible for the biosynthesis of fatty acids up to and including oleic acid. Modifying enzymes, such as the extraplastidic A12 and A15 desaturases, elongases, hydroxylases, and epoxidases, yield polyunsaturated, very long-chain, hydroxy-, and epoxy-fatty acids, respectively. Acyltransferases insert specific acyl moieties onto the glyc-

show abstract

A simple enzymatic method for the preparation of radiolabeled erucoyl-CoA and other long-chain fatty acyl-CoAs and their characterization by mass spectrometry

Cited by 106 publications

References 14 publications

Seed-Specific Over-Expression of an Arabidopsis cDNA Encoding a Diacylglycerol Acyltransferase Enhances Seed Oil Content and Seed Weight

Seed-Specific Over-Expression of an Arabidopsis cDNA Encoding a Diacylglycerol Acyltransferase Enhances Seed Oil Content and Seed Weight

Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes

Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate-Induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity

Contact Info

Product

Resources

About