Chain-shortening of erucic acid and microperoxisomal beta-oxidation in rat small intestine.

The ability of rat small intestine to chain-shorten C22:1 fatty acids was investigated. Radioactive chain-shortened products, mainly C18:1, were demonstrated in intestinal-lymph lipids after intraluminal injection of [14-14C]erucic acid. Chain-elongation to C24:1 was also observed. Adaptation to a diet containing C22:1 fatty acids (partially hydrogenated-marine-oil diet) slightly increased the percentage of chain-shortened products. Microperoxisomal beta-oxidation activity, measured as CN(-)-insensitive palmitoyl-CoA-dependent NAD+ reduction, was detected in a microperoxisome-enriched fraction from mucosal scrapings. This activity was increased 1.9-fold by a soya-bean-oil diet, and 2.7-fold by a diet containing partially hydrogenated marine oil.     

Adrenoleukodystrophy. The chain shortening of erucic acid (22:1(n-9)) and adrenic acid (22:4(n-6)) is deficient in neonatal adrenoleukodystrophy and normal in X-linked adrenoleukodistrophy skin fibroblasts

The metabolism of long chain unsaturated fatty acids was studied in cultured fibroblasts from patients with X-linked adrenoleukodystrophy (ALD) and with neonatal ALD. By using [14-14C] erucic acid (22:1(n-9)) as substrate it was shown that the peroxisomal beta-oxidation, measured as chain shortening, was impaired in cells from patients with neonatal ALD. The beta-oxidation of adrenic acid (22:4(n-6)), measured as acid-soluble products, was also reduced in the neonatal ALD cells. The peroxisomal beta-oxidation of [14-14C]erucic acid (22:1(n-9)) and [2-14C]adrenic acid (22:4(n-6)) was normal in cells from X-ALD patients. The beta-oxidation, esterification and chain elongation of [1-14C]arachidonic acid (20:4(n-6)) and [1-14C]eicosapentaenoic acid (20:5(n-3)) was normal in both X-linked ALD and in neonatal ALD. Previous studies suggest that the activation of very long chain fatty acids by a lignoceryl (24:0)-CoA ligase is deficient in X-linked ALD, while the peroxisomal beta-oxidation enzymes are deficient in neonatal ALD. The present results suggest that the peroxisomal very long-chain acyl-CoA ligase is not required for activation of unsaturated C20 and C22 fatty acids and that these fatty acids can be efficiently activated by the long chain acyl-(palmityl)-CoA ligase.     

The stimulation of erucate metabolism in isolated rat hepatocytes by rapeseed oil and hydrogenated marine oil-containing diets.

1. The metabolism of palmitate and especially of erucate was studied in hepatocytes isolated from rats fed for 3 weeks a diet containing peanut oil (diet, 1), rapeseed oil (diet 2) and partially hydrogenated marine oil (diet 3). 2. The metabolism of palmitate was not significantly influenced by the diet. The rapeseed oil diet caused 1.4 fold and 1.3 fold increase and marine oil diet 3 fold and 2.2 fold increase in the oxidation and chain-shortening respectively of [14-14C]erucic acid in isolated hepatocytes. 3. Cyanide and antimycin A did not inhibit the chain-shortening of erucate in liver cells of rats fed rapeseed oil and peanut oil. The high capacity of the chain-shortening system in hepatocytes of marine oil-fed rats was partially inhibited. 4. Inhibition of the transfer of fatty acids into the mitochondria by lowering the intracellular carnitine concentration and/or by addition of (+)-decanoyl-carnitine resulted in a very pronounced apparent stimulation of the chain-shortening of erucic acid. It is suggested that the chain-shortening system may be virtually independent of the mitochondria, unless the availability of the extramitochondria NAD+ and/or NADP+ is rate-limiting under conditions of extremely low redox potential of the mitochondria. 5. Feeding marine oil or rapeseed oil to the rats induced a 30% increase in catalase activity, a 25--30% increase in urate oxidase activity and a 50% increase in the total CoA in the liver compared to rats fed peanut oil. 6. It is suggested that the increased metabolism of erucate in hepatocytes of marine oil and rapeseed oil-fed rats may be due to the increase in ther peroxisomal beta-oxidation.     

Properties of a collagenase inhibitor partially purified from cultures of smooth muscle cells

1. The metabolism of [14-14C]erucate and [U-14C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14-14C]erucic acid by 100% and the oxidation of [U-14C]palmitic acid by 30% compared to controls. 3. The chain-shortening of erucate to C20:1 and C18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcarnitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. It is suggested that clofibrate increases the oxidation of [14-14C]erucic acid in the perfused heart by increasing the capacity for chain-shortening of [14-14C]erucate in the peroxisomal beta-oxidation system.     

The effect of feeding rats with partially hydrogenated marine oil or rapeseed oil on the chain shortening of erucic acid in perfused heart.

1. The metabolism of [14(-14)C]erucic acid and [U-14C]palmitic acid was studied in perfused hearts from rats fed diets containing hydrogenated marine oil, rapeseed oil or peanut oil for three weeks. 2. [14C]Erucic acid was shortened to [14C]eicosenoic acid (20 : 1, n -- 9) and [14C]oleic acid (18 : 1, n -- 9) in perfused rat hearts from all diet groups. The rapeseed oil diet caused a three-fold increase and the marine oil diet a four-fold increase in the amount of chain-shortened products recovered in heart lipids at the end of perfusion, compared to peanut oil diet. 3. The content of C16:1, C18:1 and C20:1 fatty acids was increased in heart lipids of rats fed hydrogenated marine oil or rapseed oil diet, compared to peanut oil diet. 4. Feeding hydrogenated marine oil or rapeseed oil to the rats induced a 85% increase in catalase activity, a 20% increase in the activity of cytochrome oxidase and a 30--40% increase in the content of total CoA in the heart compared to rats fed peanut oil diet. 5. It is suggested that [14(-14)C]erucic acid is shortened by the beta-oxidation system of peroxisomes in the heart. The increased chain shortening in the hearts from animals fed rapeseed oil or partially hydrogenated marine oil for three weeks may be an important part of an adaptation process.     

Stimulation of microperoxisomal beta-oxidation in rat heart by high-fat diets

1. Heart microperoxisomal beta-oxidation activity, measured as cyanide-insensitive palmitoyl-CoA-dependent NAD+-reduction, was detected in a microperoxisome-enriched fraction from rat myocardium. The effect on this microperoxisomal beta-oxidation of the fatty acid composition of the dietary oils was investigated. 2. Feeding 15% (w/w) high erucic acid rapeseed oil or partially hydrogenated marine oil for 3 weeks increased the microperoxisomal beta-oxidation in the heart 4-5-fold, compared to a soybean oil diet. Increasing amounts (5-30%, w/w) of partially hydrogenated marine oil in the diet led to a 3-fold increase in the microperoxisomal beta-oxidation capacity at 20% or more of this oil in the diet. 3. The activity of the microperoxisomal marker enzyme catalase followed closely the cyanide-insensitive palmitoyl-CoA-dependent NAD+-reduction, except when feeding more than 20% (w/w) partially hydrogenated marine oil where a significant decrease in the catalase activity was observed. 4. In rapeseed oil-fed animals the extent of increase of microperoxisomal beta-oxidation was directly correlated to the amount of erucic acid (22:1, n-9 cis) in the diet. 5. Feeding partially hydrogenated rapeseed oil or partially hydrogenated soybean oil resulted in activities of microperoxisomal beta-oxidation significantly lower than in the corresponding unhydrogenated oils. No significant difference could be detected between diets containing hydrogenated or unhydrogenated marine oil. 6. Addition of 5% soybean oil to the essential fatty acid-deficient, partially hydrogenated marine oil diet did not change the effect on the microperoxisomal beta-oxidation activity. 7. Clofibrate feeding increased the heart microperoxisomal beta-oxidation capacity 2.5-fold, as compared to a standard pelleted diet. 8. These findings are discussed in relation to the transient nature of the cardiac lipidosis observed with animals fed on diets rich in C22:1 fatty acids. It is concluded that the heart plays an important part in the adaptation process.     

酿酒酵母中NAD激酶同功酶对偶数位不饱和脂肪酸β-氧化的影响

ATP-NAD激酶利用ATP,催化NAD磷酸化,生成NADP,而ATP-NADH激酶则催化NAD和NADH发生磷酸化。酿酒酵母细胞内存在三种NAD激酶同功酶Utr1p、Pos5p和Yef1p,它们都是ATP-NADH激酶,对细胞内NADP(H)的供应起到重要作用。酵母偶数位双键不饱和脂肪酸的β-氧化依赖于过氧化物酶体基质内的NADPH。通过构建NAD激酶基因的单、双基因缺失株,并验证它们和对照菌株对不饱和脂肪酸的氧化、利用能力,证实NAD激酶同功酶,尤其是Pos5p,对过氧化物酶体基质内NADP(H)的供应起着重要作用,并推测NADP可以从过氧化物酶体膜外转运至过氧化物酶体基质内。     

Conclusive evidence that very-long-chain fatty acids are oxidized exclusively in peroxisomes in human skin fibroblasts

We have investigated the contribution of peroxisomes and mitochondria to the beta-oxidation of palmitate (C16:0) and cerotate (C26:0) in intact human skin fibroblasts. The oxidation of both fatty acids was found to be inhibited by rotenone plus antimycin and cyanide, respectively, although to a different extent. When 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) was used to specifically block carnitine palmitoyltransferase I, it was found that palmitate beta-oxidation was inhibited almost completely whereas cerotate beta-oxidation was not affected. Since carnitine palmitoyltransferase is essential for the oxidation of fatty acids in mitochondria this result provides conclusive evidence that oxidation of very-long-chain fatty acids is initiated in peroxisomes and not in mitochondria.     

Effects of high-fat diets on hepatic fatty acid oxidation in the rat. Isolation of rat liver peroxisomes by vertical-rotor centrifugation by using a self-generated, iso-osmotic, Percoll gradient.

1. Rat liver peroxisomal fractions were isolated in iso-osmotic Percoll gradients by using vertical-rotor centrifugation. The fractions obtained with rats given various dietary treatments were characterized. 2. The effect on peroxisomal beta-oxidation of feeding 15% by wt. of dietary fat for 3 weeks was investigated. High-fat diets caused induction of peroxisomal beta-oxidation, but diets rich in very-long-chain mono-unsaturated fatty acids produced a more marked induction. 3. Peroxisomal beta-oxidation induced by diets rich in very-long-chain mono-unsaturated fatty acids can oxidize such acids. Trans-isomers of mono-unsaturated fatty acids are oxidized at rates that are faster than, or similar to, those obtained with corresponding cis-isomers. 4. Rates of oxidation of [14-14C]erucic acid by isolated rat hepatocytes isolated from rats fed on high-fat diets increased with the time on those diets in a fashion very similar to that previously reported for peroxisomal beta-oxidation [see Neat, Thomassen & Osmundsen (1980) Biochem, J. 186, 369-371]. 5. Total liver capacities for peroxisomal beta-oxidation (expressed as acetyl groups produced per min) were estimated to range from 10 to 30% of mitochondrial capacities, depending on dietary treatment and fatty acid substrate. A role is proposed for peroxisomal beta-oxidation in relation to the metabolism of fatty acids that are poorly oxidized by mitochondrial beta-oxidation, and, in general, as regards oxidation of fatty acids during periods of sustained high hepatic influx of fatty acids.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

Effect of ischemia and reperfusion of the myocardium on in vitro beta-oxidation of fatty acids

The in vivo oxidation of perfused [14C]-labeled fatty acids has been shown to decrease dramatically in hypoxic hearts. This study addresses the influence of ischemia and reperfusion on the enzymic activities of beta-oxidation of fatty acids in mitochondria and of peroxisomal origin. The rate of beta-oxidation of fatty acids in the isolated mitochondria from myocardium of swine fed control diet declined about 20% by the ischemic insult induced by hypothermic cardioplegic arrest. Upon reperfusion, the rate of mitochondrial beta-oxidation returned to a normal level. In clofibrate-fed animals, the rate of mitochondrial beta-oxidation did not vary significantly between control, ischemic, and perfused tissues. Furthermore, neither in control nor in clofibrate-fed animals did the rates of peroxisomal beta-oxidation of fatty acids vary significantly in the ischemic or reperfused tissues as compared to that of preischemic controls. These results suggest that ischemia does not contribute to any loss of enzymic activity in beta-oxidation of fatty acid cycles either in mitochondria or peroxisomes. Furthermore, the feeding of 0.5% (w/w) clofibrate to pigs increased the rate of mitochondrial beta-oxidation of fatty acids only by 50% while that of peroxisomes increased threefold. A similar threefold increase in catalase activity was also produced by clofibrate feeding. These results suggest that the heart plays a role in the hypolipidemic action of clofibrate.     

The mechanism of the hormonal activation of respiration in isolated hepatocytes and its importance in the regulation of gluconeogenesis.

The effects of hormones on the cytochrome spectra of isolated hepatocytes were recorded under conditions of active gluconeogenesis from L-lactate. Glucagon, phenylephrine, vasopressin and valinomycin, at concentrations that caused stimulation of gluconeogenesis, increased the reduction of the components of the cytochrome bc1 complex, just as has been observed in liver mitochondria isolated from glucagon-treated rats [Halestrap (1982) Biochem. J. 204, 37-47]. The effects of glucagon and phenylephrine were additive. The time courses of the increased reduction of cytochrome c/c1 and NAD(P)H/NAD(P)+ caused by hormones, valinomycin, A23187 and ethanol were measured by dual-beam spectrophotometry and fluorescence respectively. Ethanol (14 mM) produced a substantial rise in NAD(P)H fluorescence, beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, no change in cytochrome c/c1 reduction, a 10% decrease in O2 consumption and a 60% decrease in gluconeogenesis. Glucagon, phenylephrine and vasopressin caused a substantial and transient rise in NAD(P)H fluorescence, but a sustained increase in cytochrome c/c1 reduction and the rates of O2 consumption and gluconeogenesis. The transience of the fluorescence response was greater in the absence of Ca2+, when the cytochrome c/c1 response also became transient. The fluorescence response was smaller and less transient, but the cytochrome c/c1 response was greater, in the presence of fatty acids. Both responses were greatly decreased by the presence of 1 mM-pent-4-enoate. Valinomycin (2.5 nM) caused a decrease in NAD(P)H fluorescence coincident with an increase in cytochrome c/c1 reduction and the rate of gluconeogenesis and O2 consumption. A23187 (7.5 mM) caused increases in both NAD(P)H fluorescence and cytochrome c/c1 reduction. The effects of hormones and valinomycin on the time courses of NAD(P)H fluorescence, cytochrome c/c1 reduction and light-scattering by hepatocytes were compared with those of 0.5 microM-Ca2+ or 1 nM-valinomycin on the same parameters of isolated liver mitochondria. It is concluded that hormones increase respiration by hepatocytes in a biphasic manner. An initial Ca2+-dependent activation of mitochondrial dehydrogenases rapidly increases the mitochondrial [NADH], which is followed by a volume-mediated stimulation of fatty acid oxidation and electron flow between NADH and cytochrome c. 10. Amytal (0.5 mM) was able to reverse the effects of hormones on the reduction of cytochromes c/c1 and the rates of gluconeogenesis and O2 consumption without significantly lowering tissue [ATP].(ABSTRACT TRUNCATED AT 400 WORDS)     

The alternative respiratory pathway of the yeast Candida parapsilosis: oxidation of exogenous NAD(P)H

The yeast Candida parapsilosis possesses two routes of electron transfer from exogenous NAD(P)H to oxygen. Electrons are transferred either to the classical cytochrome pathway at the level of ubiquinone through an NAD(P)H dehydrogenase, or to an alternative pathway at the level of cytochrome c through another NAD(P)H dehydrogenase which is insensitive to antimycin A. Analyses of mitoplasts obtained by digitonin/osmotic shock treatment of mitochondria purified on a sucrose gradient indicated that the NADH and NADPH dehydrogenases serving the alternative route were located on the mitochondrial inner membrane. The dehydrogenases could be differentiated by their pH optima and their sensitivity to amytal, butanedione and mersalyl. No transhydrogenase activity occurred between the dehydrogenases, although NADH oxidation was inhibited by NADP+ and butanedione. Studies of the effect of NADP+ on NADH oxidation showed that the NADH:ubiquinone oxidoreductase had Michaelis-Menten kinetics and was inhibited by NADP+, whereas the alternative NADH dehydrogenase had allosteric properties (NADH is a negative effector and is displaced from its regulatory site by NAD+ or NADP+).     

Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase

In an attempt to clarify why the brain oxidizes fatty acids poorly or not at all, the activities of beta-oxidation enzymes present in rat brain and rat heart mitochondria were measured and compared with each other. Although the apparent Km values and chain-length specificities of the brain and heart enzymes are similar, the specific activities of all but one brain enzyme are between 4 and 50% of those observed in heart mitochondria. The exception is 3-ketoacyl-CoA thiolase (EC 2.3.1.16) whose specific activity in brain mitochondria is 125 times lower than in heart mitochondria. The partially purified brain 3-ketoacyl-CoA thiolase was shown to be catalytically and immunologically identical with the heart enzyme. The low rate of fatty acid oxidation in brain mitochondria, estimated on the basis of palmitoylcarnitine-supported respiration and [1-14C]palmitoylcarnitine degradation to be less than 0.5 nmol/min/mg of protein, may be the consequence of the low activity of 3-ketoacyl-CoA thiolase. Inhibition of [1-14C]palmitoylcarnitine oxidation by 4-bromocrotonic acid proves the observed oxidation of fatty acids in brain to be dependent on 3-ketoacyl-CoA thiolase and thus to occur via beta-oxidation. Since the reactions catalyzed by carnitine palmitoyltransferase (EC 2.3.1.21) and acyl-CoA synthetase (EC 6.2.1.3) do not seem to restrict fatty acid oxidation in brain, it is concluded that the oxidation of fatty acids in rat brain is limited by the activity of the mitochondrial 3-keto-acyl-CoA thiolase.     

beta-Oxidation of the coenzyme A esters of elaidic, oleic, and stearic acids and their full-cycle intermediates by rat heart mitochondria.

beta-Oxidation rates for the CoA esters of elaidic, oleic and stearic acids and their full-cycle beta-oxidation intermediates and for the carnitine esters of oleic and elaidic acids were compared over a wide range of substrate and albumin concentrations in rat heart mitochondria. The esters of elaidic acid were oxidized at about half the rate of the oleic acid esters, while stearoyl-CoA was oxidized equally as rapid as oleoyl-CoA. The full-cycle beta-oxidation intermediates of elaidoyl-CoA (trans-16 : 1 delta 7, -14 : 1 delta 5, and -12 : 1 delta 3) were found to be oxidized at rates nearly equal to those for the corresponding intermediates of oleoyl-CoA. Therefore, after the first cycle of beta-oxidation, oleoyl-CoA and elaidoyl-CoA are oxidized at nearly equal rates. The activity of fatty acyl-CoA dehydrogenase was higher with elaidoyl-CoA and its full-cycle intermediates as substrates than with the corresponding cisisomers. It was concluded that the slower oxidation rate of elaidic acid is not due to slower oxidation of any of its full-cycle beta-oxidation intermediates, nor to slower activity of fatty acyl-CoA dehydrogenase, nor to outer mitochondrial carnitine acyltransferase. Possible explanations to account for the slower oxidation rate of elaidic acid are discussed.     

Integration of lipid utilization with Krebs cycle activity in muscle.

This essay illustrates the ways in which beta-oxidation and the citric acid cycle interact. These included: 1) competition for CoASH, 2) competition for NAD+, and 3) competition for FADH2 oxidation. By means of the above, the cell is able to maintain a precise coordination between the activation of fatty acids in the cytosol, beta-oxidation in the mitochondria, and the complete oxidation of acetyl-CoA to CO2 via the citric acid cycle throughout a wide range of energy demands and oxygen availability.     

Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in beta-oxidation convert these substrates to long-chain alpha,omega-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is omega-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C(18:1)), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to omega-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C(14:0)) much more effectively. Both enzymes, in particular CYP52A17, also oxidized omega-hydroxy fatty acids, ultimately generating the alpha,omega-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for beta-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.