Direct assay for phosphotransacetylase and acetyl-coenzyme a carboxylase by high-performance liquid chromatography

Spiegeleer, Bart De; Mannens, Geert; Slegers, Guido; Bossche, W. Van den; Claeys, A.

doi:10.1016/0003-2697(86)90609-3

Cited by 8 publications

(5 citation statements)

References 10 publications

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“…With the exception of acetyl-CoA carboxylase EC 6.4.1.2, which was assayed using the high-performance liquid chromatography (HPLC) method of De Spiegeleer et al (9), enzyme activities were determined using continuous spectrophotometric assays following the oxidation or reduction of NAD(P)(H) at 340 nm. All assays were carried out at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme

Wynn

Kendrick

Ratledge

1997

Lipids

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Sesamol, a nonoil component of sesame seed oil, inhibited growth, fatty acid synthesis, and desaturation by Mucor circinelloides in vivo. Although sesamol also inhibited the growth of other fungi and yeasts, its effect on the lipid metabolism of M. circinelloides was exceptional. An enzymological study demonstrated that sesamol affected lipid synthesis primarily by the inhibition of malic enzyme activity, thereby limiting the NADPH supply for fatty acid synthesis and desaturation. Sesamol itself had no inhibitory effect on malic enzyme activity in vitro. A metabolite of sesamol is therefore probably responsible for the in vivo effects of sesamol on lipid metabolism.

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Section: Methodsmentioning

confidence: 99%

Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme

Wynn

Kendrick

Ratledge

1997

Lipids

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“…The activity assay of ACC is based on the quantitative determination of malonyl-CoA which is formed during a certain time (De Spiegeleer et al, 1986). The enzymecatalysed reaction is stopped by boiling.…”

Section: Resultsmentioning

confidence: 99%

“…In our assay of this important enzyme, the reaction was stopped by placing the enzyme solution in boiling water for 1 min with subsequent HPLC determination of the product formed, i.e. malonyl-CoA (De Spiegeleer et al, 1986). In this paper the stability of malonyl-CoA under enzyme assay conditions is examined.…”

Section: Intr 0 D U Cti 0 Nmentioning

confidence: 99%

High performance liquid chromatography stability study of malonyl‐coenzyme A, using statistical experimental designs

Spiegeleer¹,

Sintobin

Desmet

1989

Biomedical Chromatography

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Malonyl-CoA is a biochemically important compound, formed by an acetyl-coenzyme A carboxylase catalysed reaction. The stability of this short-chain coenzyme A derivative under various experimental conditions is discussed in this article. High-performance liquid chromatography was used for the analysis of the reaction mixture because of its excellent selectivity and sufficient sensitivity. Several variables were investigated as possible stability-influencing factors: pH, magnesium and buffer concentration, reaction temperature and time. The Plackett-Burman screening design was first used for selecting the most important variables, with which a central composite design was constructed. In this way, a response surface was obtained with the percentage remaining malonyl-CoA as a function of magnesium concentration, reaction temperature and time. The usefulness of this approach is demonstrated by obtaining kinetic data from the mathematical function and by the evaluation of the stopping of reaction procedure in the activity assay of acetyl-coenzyme A carboxylase.

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“…Acetly-CoA carboxylase activity was assayed by using high-performance liquid chromatography (HPLC) to determine the amount of malonyl-CoA formed by an enzymatic reaction [ 14], 'Initial" activity was measured immediately after sample preparation without citrate preincubation, and 'citrate-activated' activity was determined after first preincubating the extract with 2 m.V/ citrate for 20 min at 30 °C [ 15], The sample was then added to a reaction mixture including 64 mM Tris (pH 7.5). 10 m.V/ trisodium citrate.…”

Section: Acelyl-coa Carboxylase Assaymentioning

confidence: 99%

Accelerated Hepatic Lipid Synthesis in Fasted Septic Rats

Kiuchi

Matsuo²,

Takeyama³

et al. 1993

Eur Surg Res

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We examined changes in the enzyme activities and metabolites related to hepatic fatty acid synthesis in fasted rats with sepsis produced by cecal ligation and puncture. Sepsis stimulated the in vivo incorporation of tritiated water into hepatic fatty acids and nonsaponifiable lipids. The activities of acetyl-CoA carboxylase, ATP-citrate lyase, and NADPH-generating enzymes (glucose-6-phosphate dehydrogenase and malic enzyme), the tissue levels of citrate and malonyl-CoA, and the dephosphorylation of carboxylase were increased in the livers of fasted septic rats compared with fasted sham-operated control rats. These results indicate that sepsis stimulated hepatic lipogenesis and sterologenesis in fasting rats. Furthermore, sepsis reduced the specific activity of hepatic mitochondrial carnitine palmitoyltransferase and raised that of glycerophos-phate acyltransferase, suggesting an increased diversion of cytosolic acyl-CoA towards esterification. These intrahepatic metabolic changes strongly suggest that sepsis causes anabolic action on hepatic lipid metabolism.

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Direct assay for phosphotransacetylase and acetyl-coenzyme a carboxylase by high-performance liquid chromatography

Cited by 8 publications

References 10 publications

Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme

Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme

High performance liquid chromatography stability study of malonyl‐coenzyme A, using statistical experimental designs

Accelerated Hepatic Lipid Synthesis in Fasted Septic Rats

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