Inhibitory effect of very-long-chain monounsaturated fatty-acyl-CoAs on the elongation of long-chain fatty acid in swine cerebral microsomes

Characteristics of condensation and overall elongation of very-long-chain fatty-acyl-CoAs in swine cerebral microsomes were studied using radio high-performance liquid chromatography (RHPLC) and gas chromatography-mass spectrometry (GC-MS). The monounsaturated fatty-acyl-CoA depressed both the condensation and overall elongation activities of endogenous substrates and also of exogenous saturated fatty-acyl-CoA. The extent of the decrease of the elongation activity was dependent on the concentration and the chain length of the exogenous fatty-acyl-CoAs. The dependence of the condensation activity of monounsaturated fatty-acyl-CoA on the concentration of malonyl-CoA suggested that the non-Michaelis-Menten type kinetics was dominant for oleoyl-CoA, however, a normal kinetic pattern was obtained for endogenous palmitoyl-CoA and arachidonoyl-CoA with Km = 37 microM to malonyl-CoA. The condensation activity for icosanoyl-CoA (20:0-CoA) was inhibited by icosenoyl-CoA (20:1-CoA) in a non-competitive manner, which suggested that the condensation enzyme, or at least the active center of the enzyme for icosenoyl-CoA, was different from that for icosanoyl-CoA.     

Comparison between condensation and overall chain elongation of arachidoyl-CoA and arachidonoyl-CoA in swine cerebral microsomes

The condensation and overall elongation products of exogenous arachidoyl-CoA (20:0-CoA) and endogenous fatty acids in swine cerebral microsomes were detected by radio gas chromatography. In addition, the condensation products with malonyl-CoA as substrate were analyzed by radio high-performance liquid chromatography. Three main condensation products were detected; the overall elongation products of exogenous 20:0-CoA were 22:0 and 24:0, and those of endogenous substrates were 18:0, 22:4, and 24:4. The yield was estimated for the conversion of 3-ketoacyl-CoAs to the corresponding saponification products (methyl ketones or R-2-one; e.g., 2-heptadecanone = 17:0-2-one); these products were identified in the preceding paper (S. Yoshida and M. Takeshita (1987) Arch. Biochem. Biophys. 254, 170-179). The extraction of R-2-one by hexane depended on the acyl chain length. The yield of 2-heneicosanone (21:0-2-one) detected by radio gas chromatography was 80% whereas the yields of 17:0-2-one and 2-heneicosatetraenone (21:4-2-one) from the corresponding 3-ketoacyl-CoAs were 56 and 48%, respectively. A quantitative comparison was performed for the condensation and overall elongation activity; it was noticed that the condensation activity for the system which simultaneously produced two elongation products was nearly the same as that of the corresponding overall elongation activity. This result suggests that the condensation step may be at least one of the rate-limiting steps in the overall elongation of very-long-chain fatty acyl-CoA.     

Arachidoyl- and arachidonoyl-CoA elongation mechanism in swine cerebral microsomes

Long-chain saturated and polyunsaturated fatty acyl-CoA elongations were studied in swine cerebral microsomes. The elongation of endogenous palmitoyl-CoA to stearate was highly active in both cerebral and liver microsomes, whereas those of arachidoyl-CoA (20:0-CoA) and endogenous arachidonoyl-CoA (20:4-CoA) were high in cerebral microsomes, but negligible in liver microsomes. The elongation of 22:4 to 24:4 was also observed in cerebral microsomes. Both NADPH and NADH at 500 microM were effective in elongation of 16:0-, 20:0- and 20:4-CoA, whereas NADPH was more effective in elongation of 22:4 to 24:4 than NADH. The incorporation of deuterium atoms to the elongated product was detected by the technique of mass fragmentography when the NADPH-dependent elongations of 20:0-CoA and 20:4-CoA were performed in 2H2O medium upon cerebral microsomes. The number of incorporated deuterium atoms into 22:0 elongated from 20:0-CoA was mainly two, and that into 22:4 elongated from 20:4-CoA was mainly three. These results indicated that part of hydrogens in elongated arachidoyl- and arachidonoyl-CoA were transferred from NADPH.     

Stereospecific incorporation of hydrogens from NADPH in elongation of very long fatty acyl-CoA by swine cerebral microsomes

The elongation of arachidoyl-CoA by swine cerebral microsomes resulted in the production of behenic acid (22:0) and lignoceric acid (24:0) concomitantly. When 4S-[4-²H₁]NADPH was used for the elongation of arachidoyl-CoA, the incorporation of two deuterium atoms into 22:0 was observed by the technique of mass fragmentography. Furthermore, the incorporation of four deuterium atoms into 24:0 was also detected. On the other hand, when 4R-[4-²H₁]NADPH was used, no deuterium was incorporated into the elongated products.     

Effect of fatty-acyl-CoAs on the elongation of saturated fatty acid in porcine aorta microsomes

The microsomal elongation system from porcine aorta for longchain fatty-acyl-CoAs was investigated. Palmitoleoyl-CoA (16:1-CoA), oleoyl-CoA (18:1-CoA), and eicosenoyl-CoA (20:1-CoA) remarkably depressed the elongation activity for 16:0-CoA in aorta microsomes by 44.8, 52.4, and 43.7% of the control activity, respectively. Saturated and polyunsaturated fatty-acyl-CoAs had little effect on the 16:0-CoA elongation activity. These results indicate that monounsaturated long-chain fatty acyl-CoAs can regulate the synthesis of saturated fatty acids in the vessel walls.     

Effect of phospholipase A2 and free fatty acids on lipid-protein interactions in long- and very-long-chain fatty acyl-CoA elongation enzyme systems of brain microsomes

The elucidation of the mechanism of phospholipase A2-induced inactivation of the condensation enzyme provided evidence concerning the important role of lipid-enzyme interactions in maintaining the condensation activity in swine cerebral microsomes. A quantitative analysis of fatty acid release by phospholipase A2 from the microsomal membrane revealed that only 5 nmol of free fatty acid per mg microsomal protein was released, including oleic acid and arachidonic acid, by treatment with 0.4 unit of phospholipase A2 per mg microsomal protein for 15 s at 23 degrees C. Under these conditions, the condensation activity for endogenous 16:0-CoA and 20:4-CoA decreased to half and that for exogenous 20:0-CoA decreased to 75%. However, the addition of free fatty acids and lysophospholipids or a mixture of them at 5-10 nmol/mg protein did not change the condensation activity for endogenous 16:0-CoA and 20:4-CoA, or for exogenous 20:0-CoA. These results indicated that phospholipase A2 inhibited the condensation activity by acting directly on phospholipids that are indispensable to maintaining the function of the condensation enzyme. The Arrhenius plot for the condensation of endogenous 16:0-CoA showed a break at around 16 degrees C, whereas no break of the plot was observed for the condensation of 20:0-CoA and 20:4-CoA. The activation energy for the condensation of 16:0-CoA and 20:4-CoA was decreased by the addition of free fatty acids such as oleic acid and stearic acid, with disappearance of the Arrhenius break for 16:0-CoA condensation, whereas the activation energy for the condensation of 20:0-CoA was not changed. These results suggest that the type of lipid-protein interaction in the condensation enzyme for 20:0-CoA is different from that for 16:0-CoA and 20:4-CoA.     

Characterization of fatty acid elongation system in porcine neutrophil microsomes

Microsomes purified from porcine neutrophils containing the fatty acid chain-elongation system for long- and very-long-chain fatty acyl-CoAs, and several enzymatic characters for the elongation of palmitoyl-CoA (16:0-CoA) and arachidoyl-CoA (20:0-CoA) were examined. The heat-inactivation profile for the elongation of 16:0-CoA was different from that of 20:0-CoA, suggesting the presence of different enzyme systems for palmitoyl-CoA and arachidoyl-CoA. Contrary to the elongation system of brain microsomes, the successive synthesis of lignoceric acid (24:0) from 20:0-CoA at 60 microM was not prominent under normal conditions in the neutrophil microsomes. The synthesis of behenic acid (22:0) was slightly inhibited by 0.5 mM N-ethylmaleimide (NEM) present in the assay mixture, whereas the pre-treatment of microsomes with 0.5 mM NEM largely inhibited the synthesis of 22:0 from 20:0-CoA. The synthesis of 24:0, however, was enhanced by 0.5 mM NEM in the elongation of 20:0-CoA and the rate of 24:0 synthesis became dominant over the synthesis of 22:0. These results suggested that the elongation enzyme for very-long-chain fatty acyl-CoA, especially for 20:0-CoA elongation to 22:0 in the neutrophil microsomes contained NEM-sensitive sulfhydryl groups in the active center and the mechanism for the synthesis of 24:0 through successive elongation from 20:0-CoA was different from that of 22:0, as the former was enhanced by NEM whereas the latter was strongly inhibited.     

Presence of three B-type cytochromes in swine cerebral microsomes

In swine cerebral microsomes purified with sucrose density gradient and glycerol-cholate gradient centrifugations, it was observed that a new b-type cytochrome which had alpha-peak at 560 nm and Soret peak at 428 nm at 23 degrees C was reduced preferentially by anaerobic NADPH in the presence of cyanide. The b5-type cytochromes were reduced completely by both NADH and NADPH anaerobically. Three b-type cytochromes were partially purified into two b-type, spectroscopically distinct from each other, and the new b-type (b560-5) cytochromes.     

Substrates and products of fatty acid elongation in mouse brain microsomes]

In brain microsomes, palmitate and stearate elongation involve a membrane lipid-bound substrate. After elongation by malonyl-CoA, acyl-products are partially bound to proteins. Acyl-proteins are not found when endogenous fatty acid elongation takes place. In the dysmyelinating Quaking mouse mutants, "stearyl-membrane" substrate formation is normal; thus, the deficiency observed in very long chain fatty acid formation is not due to a lack in substrate formation.     

Metabolism of saturated and polyunsaturated very-long-chain fatty acids in fibroblasts from patients with defects in peroxisomal beta-oxidation.

The metabolism of [1-14C]lignoceric acid (C24:0) and [1-14C]tetracosatetraenoic acid (C24:4, n-6) was studied in normal skin fibroblast cultures and in cultures from patients with defects in peroxisomal beta-oxidation (but normal peroxisomal numbers). Cells from X-linked adrenoleukodystrophy (ALD) patients with a presumed defect in a peroxisomal acyl-CoA synthetase, specific for fatty acids of carbon chain lengths greater than 22 (very-long-chain fatty acids; VLCFA), showed a relatively normal production of radiolabelled CO2 and water-soluble metabolites from [1-14C]C24:0. However, the products of synthesis from acetate de novo (released by beta-oxidation), i.e. C16 and C18 fatty acids, were decreased, and carbon chain elongation of the fatty acid was increased. In contrast, cell lines from two patients with an unidentified lesion in peroxisomal beta-oxidation (peroxisomal disease, PD) showed a marked deficiency in CO2 and water-soluble metabolite production, a decreased synthesis of C16 and C18 fatty acids and an increase in carbon chain elongation. The relatively normal beta-oxidation activity of ALD cells appears to be related to low uptake of substrate, as a defect in beta-oxidation is apparent when measurements are performed on cell suspensions under high uptake conditions. Oxidation of [1-14C]C24:4 was relatively normal in ALD cells and in the cells from one PD patient but abnormal in those from the other. Our data suggest that, despite the deficiency in VLCFA CoA synthetase, ALD cells retain a near normal ability to oxidize both saturated and polyunsaturated VLCFA under some culture conditions. However, acetate released by beta-oxidation of the saturated VLCFA and, to a much lesser degree, the polyunsaturated VLCFA, appears to be used preferentially for the production of CO2 and water-soluble products, and acetate availability for fatty acid synthesis in other subcellular compartments is markedly decreased. It is likely that the increased carbon chain elongation of the saturated VLCFA which is also observed reflects the increased availability of substrate (C24:0) and/or an increase in microsomal elongation activity in ALD cells.     

Fat metabolism in higher plants. The elongation of saturated and unsaturated acyl-CoAs by a stromal system from isolated spinach chloroplasts.

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.     

Concomitant decrease in the elongation and condensation activities of very-long chain fatty acyl-CoA in jimpy mouse

Overall elongation and condensation of long-chain and very-long-chain fatty acids have been studied in the brain microsomes of jimpy mice. Both the elongation and condensation activities with stearoyl (18:0)-, oleoyl (18:1)- and arachidoyl (20:0)-CoA were severely diminished in jimpy brain, but the decrease in the activity with the exogenous palmitoyl (16:0)-CoA was less pronounced. The decrease in the elongation and condensation reactions with endogenous palmitic and arachidonic (20:4) acids was not distinct in the mutant. The decrease in the activity of condensation reaction may be responsible for the reduced rate of overall fatty acid elongation.     

Inhibition of saturated very-long-chain fatty acid biosynthesis by mefluidide and perfluidone,selective inhibitors of 3-ketoacyl-CoA synthases

The trifluoromethanesulphonanilides mefluidide and perfluidone are used in agriculture as plant growth regulators and herbicides. Despite the fact that mefluidide and perfluidone have been investigated experimentally for decades, their mode of action is still unknown. In this study, we used a cascade approach of different methods to clarify the mode of action and target site of mefluidide and perfluidone. Physiological profiling using an array of biotests and metabolic profiling in treated plants of Lemna paucicostata suggested a common mode of action in very-long-chain fatty acid (VLCFA) synthesis similar to the known 3-ketoacyl-CoA synthase (KCS) inhibitor metazachlor. Detailed analysis of fatty acid composition in Lemna plants showed a decrease of saturated VLCFAs after treatment with mefluidide and perfluidone. To study compound effects on enzyme level, recombinant KCSs from Arabidopsis thaliana were expressed in Saccharomyces cerevisiae. Enzyme activities of seven KCS proteins from 17 tested were characterized by their fatty acid substrate and product spectrum. For the KCS CER6, the VLCFA product spectrum in vivo, which consists of tetracosanoic acid, hexacosanoic acid and octacosanoic acid, is reported here for the first time. Similar to metazachlor, mefluidide and perfluidone were able to inhibit KCS1, CER6 and CER60 enzyme activities in vivo. FAE1 and KCS2 were inhibited by mefluidide only slightly, whereas metazachlor and perfluidone were strong inhibitors of these enzymes with IC₅₀ values in μM range. This suggests that KCS enzymes in VLCFA synthesis are the primary herbicide target of mefluidide and perfluidone.     

Activation of synthesis of hexacosenoic acid by sulfhydryl reagents in swine cerebral microsomes

The elongation of icosenoyl-CoA (20:1-CoA) in swine cerebral microsomes resulted in the synthesis of docosenoic acid (22:1) and tetracosenoic acid (24:1), but the synthesis of hexacosenoic acid (26:1) was negligible. In contrast, in the presence of sulfhydryl reagents (0.6 mM N-ethylmaleimide [NEM] or 0.3 mM p-chloromercuriphenylsulfonic acid [PCMPS]) the synthesis of 26:1 was remarkably enhanced. We suggest that the synthesis of 26:1 from 20:1-CoA was more enhanced by NEM or PCMPS as a result of activation of the condensation step in the elongation of 24:1 (intermediate) to 26:1.     

Interaction of amphiphilic substrates (acyl-CoAs) and their metabolites (free fatty acids) with microsomes from mouse sciatic nerves

We have measured the partition of stearoyl-CoA and oleoyl-CoA between an aqueous phase and the microsomes from mouse sciatic nerves. A method of microultracentrifugation was used which allowed us to study separately the aqueous phase and the biological membranes. We observed that the partition is dependent upon the amount of acyl-CoAs and membrane proteins but seems to be independent of time. A theoretical analysis of these data allowed interpretation of the binding and release in terms of acyl-CoA surface density in the vesicles. We have also analyzed the fate of the membrane-bound acyl-CoAs. We show that, whereas the apparent partition does not seem to vary, the hydrolysis of the membrane-bound acyl-CoAs followed by the release of free fatty acids from the membrane leads to a modification of the partition of acyl-CoAs between the membrane and the aqueous phase. We propose that there is a constant partition of the aliphatic chains (acyl-CoAs + free fatty acids).     

Separate peroxisomal oxidases for fatty acyl-CoAs and trihydroxycoprostanoyl-CoA in human liver

Fatty acyl-CoAs as well as the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids are beta-oxidized in peroxisomes. The first reaction of peroxisomal beta-oxidation is catalyzed by acyl-CoA oxidase. We recently described the presence of two fatty acyl-CoA oxidases plus a trihydroxycoprostanoyl-CoA oxidase in rat liver peroxisomes (Schepers, L., P. P. Van Veldhoven, M. Casteels, H. J. Eyssen, and G. P. Mannaerts. 1990. J. Biol. Chem. 265: 5242-5246). We have now developed methods for the measurement of palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase in human liver. The activities were measured in livers from controls and from three patients with peroxisomopathies. In addition, the oxidase activities were partially purified from control livers by ammonium sulfate fractionation and heat treatment, and the partially purified enzyme preparation was subjected to chromatofocusing, hydroxylapatite chromatography, and gel filtration. In earlier experiments this allowed for the separation of the three rat liver oxidases. The results show that human liver, as rat liver, contains a separate trihydroxycoprostanoyl-CoA oxidase. In contrast to the situation in rat liver, no conclusive evidence was obtained for the presence of two fatty acyl-CoA oxidases in human liver. Our results explain why bile acid metabolism is normal in acyl-CoA oxidase deficiency, despite a severely disturbed peroxisomal fatty acid oxidation and perhaps also why, in a number of other cases of peroxisomopathy, di- and trihydroxycoprostanic acids are excreted despite a normal peroxisomal fatty acid metabolism.