Inhibitor Of acyl-CoA Synthetase Research Articles

The inhibition of long-chain fatty acyl-CoA synthetase by enoximone in rat heart mitochondria.

The mechanism by which enoximone, a reported phosphodiesterase inhibitor, inhibits the oxidation of long-chain fatty acids was studied in isolated rat heart mitochondria using a series of 14C-labeled substrates. Enoximone decreased palmitate oxidation in a time- and concentration-dependent manner. Fifty percent inhibition of palmitate oxidation was achieved with 250 microM of enoximone. In contrast to its effect on palmitate, enoximone (250 microM) increased octanoate oxidation by 30%, whereas pyruvate oxidation was unaffected by enoximone. At that dose there was no effect on the oxidation of palmitoyl-CoA and palmitoyl carnitine. The degree of palmitate oxidation inhibited by enoximone was parallel to the inhibition of acyl-CoA synthetase in both rat heart mitochondria and microsomes. These results suggest that enoximone is a reversible inhibitor of long-chain fatty acyl-CoA synthetase. Moreover, the reaction, which is catalyzed by this enzyme, is a rate-limiting step in the pathway of fatty acid oxidation in rat heart mitochondria.

The Acyl-CoA Synthetase Inhibitor Triacsin C Enhanced Eicosanoid Release in Leukocytes

Triacsin C was previously reported to inhibit long-chain acyl-CoA synthetase and to reduce the production of acyl-CoA in rat neutrophils dose- and time-dependently. We found that preincubation with triacsin C inhibited the incorporation of labeled arachidonic acid into rat neutrophils. Furthermore, when triacsin C-treated neutrophils were stimulated with the Ca-ionophore A23187, they released an increased amount of eicosanoids into the culture medium. These results indicate that triacsin C suppressed acylation of arachidonic acid, which resulted in an increase in its metabolites.

The acyl-CoA synthetase inhibitor triacsin C enhanced eicosanoid release in leukocytes.

Triacsin C was previously reported to inhibit long-chain acyl-CoA synthetase and to reduce the production of acyl-CoA in rat neutrophils dose- and time-dependently. We found that preincubation with triacsin C inhibited the incorporation of labeled arachidonic acid into rat neutrophils. Furthermore, when triacsin C-treated neutrophils were stimulated with the Ca-ionophore A23187, they released an increased amount of eicosanoids into the culture medium. These results indicate that triacsin C suppressed acylation of arachidonic acid, which resulted in an increase in its metabolites.

Inhibition of long-chain ACYL-CoA synthetase by the peroxisome proliferator perfluorodecanoic acid in rat hepatocytes

Perfluorodecanoic acid (PFDA) is a potent peroxisome proliferator and is known to affect hepatic lipid metabolism in rats. The effects of PFDA on fatty acid utilization were examined in isolated rat hepatocyte suspensions and in rat liver mitochondria and microsomes. PFDA inhibited the oxidation of palmitic acid but not octanoic or pyruvic acids when hepatocytes were incubated with 1 mM PFDA. At this PFDA concentration the esterification of palmitic acid into triacylglycerols was also reduced. The activity of long-chain acyl-CoA synthetase (ACS), an enzyme essential for both oxidation and esterification of fatty acids, was reduced in hepatocytes incubated with 1 mM PFDA. Carnitine palmitoyltransferase (CPT), an important enzyme for the oxidation of long-chain fatty acids, was not altered in hepatocytes incubated with this PFDA concentration. In rat liver mitochondria, palmitate oxidation and ACS activity were reduced significantly (P<0.01) at a PFDA concentration that had no effect on CPT activity. The inhibition of ACS by PFDA was similar in liver mitochondria and microsome preparations. In mitochondria incubated with PFDA, the inhibition of ACS appears to be noncompetitive for the substrates palmitic acid and CoA. However, the ACS inhibition by PFDA appeared to be competitive for the ATP binding site of the enzyme. Several chain length perfluorinated fatty acids were examined for their ability to inhibit mitochondrial ACS. Short-chain perfluorinated fatty acids (perfluoroproprionic and -butyric acid) did not inhibit ACS activity. However, medium-chain perfluorinated acids (perfluorooctanoic, -ananoic and -decanoic acid) were found to be potent inhibitors of ACS in isolated mitochondria. Whether ACS inhibition is causally related to PFDA-induced peroxisome proliferation and altered lipid metabolism seen in vivo is yet to be determined.

Evidence for an essential role of long chain acyl-CoA synthetase in animal cell proliferation: Inhibition of long chain acyl-CoA synthetase by triacsins caused inhibition of Raji cell proliferation

Triacsins A, B, C, and D are new inhibitors of long chain acyl-CoA synthetase (EC 6.2.1.3) and possess different inhibitory potencies against the enzyme (Tomoda, H., Igarashi, K., and Omura, S. (1987) Biochim. Biophys. Acta 921, 595-598). Acyl-CoA synthetase activity in the membrane fraction of Raji cells was also inhibited by triacsins. The same hierarchy of inhibitory potency as that against the enzyme from other sources, triacsin C greater than triacsin A much greater than triacsin D greater than or equal to triacsin B, was observed. When Raji cells were cultivated in the presence of triacsins, cell proliferation was inhibited in a dose-dependent fashion. The drug concentrations required for 50% inhibition of cell growth at day 2 were calculated to be 1.8 microM for triacsin A, much greater than 20 microM for triacsin B, 1.0 microM for triacsin C, and much greater than 15 microM for triacsin D, demonstrating a hierarchy for inhibitory potency of triacsins similar to that against the acyl-CoA synthetase activity. To understand the role of long chain acyl-CoA synthetase in animal cells, the effect of triacsins on the lipid metabolism of Raji cells was studied. When intact Raji cells were incubated with [14C]oleate in the presence of individual triacsins, the incorporation of [14C]oleate into each of the lipid fractions such as phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol was inhibited to an analogous extent. A common hierarchy, triacsin C greater than triacsin A much greater than triacsin D greater than triacsin B, was shown for the inhibition in each synthesis of the three lipids, which was identical with that for acyl-CoA synthetase. These findings indicate that the inhibition of acyl-CoA synthetase is well correlated with the inhibition of lipid synthesis. Taken together, the data strongly suggest that the inhibition of acyl-CoA synthetase by triacsins leads to the inhibition of lipid synthesis and eventually to the inhibition of proliferation of Raji cells.

Fatty acyl-coenzyme a is required for budding of transport vesicles from Golgi cisternae

We describe a new role for fatty acylation. Conditions were established under which vesicular transport from the cis to the medial Golgi compartment in vitro depends strongly upon the addition of a fatty acylcoenzyme A, e.g., palmitoyl-CoA. Using an inhibitor of long-chain acyl-CoA synthetase, we demonstrate that the fatty acid has to be activated by CoA to stimulate transport. A nonhydrolyzable analog of palmitoyl-CoA competitively inhibits transport. Electron microscopy and biochemical studies show that fatty acyl-CoA is required for budding of (non-clathrin-) coated transport vesicies from Golgi cisternae and that budding is inhibited by the nonhydrolyzable analog.

Triacsin C: A differential inhibitor of arachidonoyl-CoA synthetase and nospecific long chain acyl-CoA synthetase,

Triacsins A,B,C, and D are newly discovered compounds isolated from the culture filtrate of streptomyces which are known to inhibit nonspecific long chain acyl-CoA synthetase (EC 6.2.1.3.). These inhibitors have not been previously studied with regard to their effects on arachidonoyl-CoA synthetase, an enzyme which specifically utilizes arachidonate and other icosanoid precursor fatty acids. To explore his question, we used triacsin C, a potent inhibitor of the nonspecific acyl-CoA synthetase. Triacsin C was found to inhibit the action of arachidonoyl-CoA synthetase and the nonspecific enzyme in sonicates of HSDM 1C 1 mouse fibrosarcoma cells. Importantly, however, the triacsin concentration and length of pre-incubation with the enzymes could be adjusted to almost completely inhibit (>80%) the nonspecific long chain acyl CoA-synthetase, with less than 20% inhibition of arachidonoyl-CoA synthetase. Using intact cultured cells exposed to 1 ug/ml traicsin for up to 15 minutes, we unexpectedly observed preferential inhibition of arachidonoyl-CoA synthetase activity. In intact cell studies, arachidonoyl-CoA synthetase was inhibited > 90%, with 55–60% inhibition of the nonspecific acyl-CoA synthetase. As additional evidence of its inhibition of acyl-CoA synthetase enzymes in intact cells, triacsin c inhibited both fatty acid uptake into cells and icosanoid production, metabolic processes which in certain cell types appear to be dependent on acyl-CoA synthetase activity. Thus, triacsin C is a novel inhibitor which can alter the fatty metabolism of intact cells. This compound can be of significant value in determining the specific cellular functions of the two acyl-CoA synthetase enzymes.

Influence of 5-tridecylpyrazole-3-carboxylic acid, a new hypolipidaemic agent, on cholesteryl ester formation in rabbit intestinal mucosa.

The comparative effects of 5-tridecylpyrazole-3-carboxylic acid (TDPC), beta-sitosterol and melinamide on the esterification of cholesterol (CH) have been investigated in rabbit intestinal microsomes and cytosol in-vitro. The three agents did not show an effect on cholesteryl ester formation by cholesterol esterase (CEase). TDPC and beta-sitosterol did not affect cholesteryl oleate formation from oleoyl CoA by microsomal acyl CoA:cholesterol acyltransferase (ACAT), whereas melinamide significantly inhibited cholesteryl oleate formation. TDPC significantly inhibited the incorporation of oleic acid into cholesteryl oleate, which is associated with acyl CoA synthetase (ACS) plus ACAT in mucosal microsomes, at a concentration of 20-100 microM. On the other hand, 5-tridecylpyrazole-3-carbinol (TDPC-OH) a congener of TDPC, and beta-sitosterol did not show any effect. From these results, it is demonstrated that carboxylic moiety of TDPC is necessary to inhibit ACS in-vitro. According to the kinetic analytical results, it is suggested that TDPC acts as a competitive inhibitor of ACS. These results suggest that the inhibitory effect of TDPC on cholesteryl ester formation may be mediated by an inhibition of ACS activity. It is apparent from the data presented that there are substantial differences between TDPC, beta-sitosterol and melinamide with respect to their action on cholesteryl ester formation in rabbit intestinal mucosa.

Inhibition of acyl-CoA synthetase by triacsins

Triacsin A, 1-hydroxy-3-( E, E-2'4'-undecadienylidine) triazene and triacsin C, 1-hydroxy-3-( E, E, E-2',4',7'-undecatrienylidine) triazene are potent inhibitors of acyl-CoA synthetase (EC 6.2.1.3). The concentrations of triacsin A required for 50% inhibition of acyl-CoA synthetase from Pseudomonas aeruginosa and from rat liver are 17 and 18 μM, and those of triacsin C are 3.6 and 8.7 μM, respectively. Kinetic analysis indicates that inhibition of triacsin A is non-competitive with respect to the two substrates ATP and coenzyme A, but is competitive with respect to long-chain fatty acids. The apparent K i value is 8.97 μM when oleic acid is used as substrate. Acid hydrolysis of triacsins results in corresponding polyenic aldehydes with no activity. This suggests that the N-hydroxytriazene moiety is essential for inhibitory activity against acyl-CoA synthetase.