Long-chain Acyl Coenzyme A Research Articles

Fatty acyl-coenzyme A activates mitochondrial division through oligomerization of MiD49 and MiD51.

Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl-coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, which activates their ability to stimulate the DRP1 GTPase. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knockdown effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with BSA-bound oleic acid, which causes increased LCACA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty-acid-induced mitochondrial division. Finally, MiD49 or MiD51 oligomers synergize with Mff, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation.

Structural basis of the activation of TRPV5 channels by long-chain acyl-Coenzyme-A

Long-chain acyl-coenzyme A (LC-CoA) is a crucial metabolic intermediate that plays important cellular regulatory roles, including activation and inhibition of ion channels. The structural basis of ion channel regulation by LC-CoA is not known. Transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6) are epithelial calcium-selective ion channels. Here, we demonstrate that LC-CoA activates TRPV5 and TRPV6 in inside-out patches, and both exogenously supplied and endogenously produced LC-CoA can substitute for the natural ligand phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in maintaining channel activity in intact cells. Utilizing cryo-electron microscopy, we determined the structure of LC-CoA-bound TRPV5, revealing an open configuration with LC-CoA occupying the same binding site as PI(4,5)P2 in previous studies. This is consistent with our finding that PI(4,5)P2 could not further activate the channels in the presence of LC-CoA. Our data provide molecular insights into ion channel regulation by a metabolic signaling molecule.

Effect of Dendrobium nobile Lindl. alkaloids on myocardial lipid metabolism during cardiopulmonary bypass ischemia-reperfusion in dogs

Objective: To explore the effects and mechanisms of Dendrobium nobile Lindl. alkaloids (DNLA) on myocardial lipid metabolism during ischemia-reperfusion in dogs undergoing cardiopulmonary bypass (CPB). Methods: Twenty-four healthy hybrid dogs, half male and half female, were randomly divided into sham group, model group, solvent control group and treatment group (DNLA, 6 mg/kg) (n=6), all of which were established with CPB. Except for the sham group, the aorta of the other groups was occluded for 60 min and then reopened. The uptake rate of free fatty acids, the concentration of long-chain acyl coenzyme A (LCACoA), mRNA and protein expression of fatty acid translocase enzyme/CD36 (FAT/CD36) in myocardial tissue and the cardiac function indexes were measured at 4 time points: before cardiopulmonary bypass (T1), 15 min (T2), 60 min (T3), and 90 min (T4) after reperfusion in each group. Results: Before CPB, there were no statistically significant differences in the uptake rate of free fatty acids, the concentration of LCACoA and mRNA expression of FAT/CD36 in myocardial tissue in each group (P>0.05). After the opening of the aorta, the above indexes in model group [(35.8±4.7)%, (8.55±1.51) nmol/g, 3.23±0.68] and treatment group [(27.4±2.7)%, (6.10±1.38) nmol/g, 2.20±0.56] were higher than those in sham group [(19.6±3.9)%, (4.16±0.81)nmol/g, 1.19±0.52], which were the highest at T2, and then gradually decreased (all P<0.05). Compared with the model group, the increase of above indicators in the treatment group was significantly lower at T2 (all P<0.05). Before CPB, there was no statistically significant differences in cardiac function indexes [left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and±dp/dtmax] among the groups (P>0.05). After the aorta was opened, the above indexes in model group [(76.5±9.1) mmHg, (31.1±2.9) mmHg, (1.2±0.4) mmHg/ms, (-0.9±0.1) mmHg/ms] and treatment group [(92.9±8.7) mmHg, (25.3±3.6) mmHg, (1.8±0.4) mmHg/ms, (-1.3±0.1) mmHg/ms] were lower than those in sham group [(165.5±12.9) mmHg, (6.5±0.5) mmHg, (3.3±0.6) mmHg/ms, (-2.9±0.3) mmHg/ms] (all P<0.05), but the impairment degree of cardiac function indicators in treatment group was significantly lower than that those in model group (all P<0.05). Conclusion: During CPB in dogs, DNLA can inhibit the abnormal expression of FAT/CD36, decrease the uptake of free fatty acids, and reduce the abnormal accumulation of LCACoA in myocardium,thereby alleviating the myocardial injury after ischemia-reperfusion.

Preservation of Acyl Coenzyme A Attenuates Pathological and Metabolic Cardiac Remodeling Through Selective Lipid Trafficking.

Metabolic remodeling in heart failure contributes to dysfunctional lipid trafficking and lipotoxicity. Acyl coenzyme A synthetase-1 (ACSL1) facilitates long-chain fatty acid (LCFA) uptake and activation with coenzyme A (CoA), mediating the fate of LCFA. The authors tested whether cardiac ACSL1 overexpression aids LCFA oxidation and reduces lipotoxicity under pathological stress of transverse aortic constriction (TAC). Mice with cardiac restricted ACSL1 overexpression (MHC-ACSL1) underwent TAC or sham surgery followed by serial in vivo echocardiography for 14 weeks. At the decompensated stage of hypertrophy, isolated hearts were perfused with 13C LCFA during dynamic-mode 13C nuclear magnetic resonance followed by in vitro nuclear magnetic resonance and mass spectrometry analysis to assess intramyocardial lipid trafficking. In parallel, acyl CoA was measured in tissue obtained from heart failure patients pre- and postleft ventricular device implantation plus matched controls. TAC-induced cardiac hypertrophy and dysfunction was mitigated in MHC-ACSL1 hearts compared with nontransgenic hearts. At 14 weeks, TAC increased heart weight to tibia length by 46% in nontransgenic mice, but only 26% in MHC-ACSL1 mice, whereas ACSL1 mice retained greater ejection fraction (ACSL1 TAC: 65.8±7.5%; nontransgenic TAC: 45.9±7.3) and improvement in diastolic E/E'. Functional improvements were mediated by ACSL1 changes to cardiac LCFA trafficking. ACSL1 accelerated LCFA uptake, preventing C16 acyl CoA loss post-TAC. Long-chain acyl CoA was similarly reduced in human failing myocardium and restored to control levels by mechanical unloading. ACSL1 trafficked LCFA into ceramides without normalizing the reduced triglyceride storage in TAC. ACSL1 prevented de novo synthesis of cardiotoxic C16- and C24-, and C24:1 ceramides and increased potentially cardioprotective C20- and C22-ceramides post-TAC. ACLS1 overexpression activated AMP activated protein kinase at baseline, but during TAC, prevented the reduced LCFA oxidation in hypertrophic hearts and normalized energy state (phosphocreatine:ATP) and consequently, AMP activated protein kinase activation. This is the first demonstration of reduced acyl CoA in failing hearts of humans and mice, and suggests possible mechanisms for maintaining mitochondrial oxidative energy metabolism by restoring long-chain acyl CoA through ASCL1 activation and mechanical unloading. By mitigating cardiac lipotoxicity, via redirected LCFA trafficking to ceramides, and restoring acyl CoA, ACSL1 delayed progressive cardiac remodeling and failure.

Updating the effects of fatty acids on skeletal muscle

In this review we updated the fatty acid (FA) effects on skeletal muscle metabolism. Abnormal FA availability induces insulin resistance and accounts for several of its symptoms and complications. Efforts to understand the pathogenesis of insulin resistance are focused on disordered lipid metabolism and consequently its effect on insulin signaling pathway. We reviewed herein the FA effects on metabolism, signaling, regulation of gene expression and oxidative stress in insulin resistance. The elevated IMTG content has been associated with increased intracellular content of diacylglycerol (DAG), ceramides and long-chain acyl-coenzyme A (LCA-CoA). This condition has been shown to promote insulin resistance by interfering with phosphorylation of proteins of the insulin pathway including insulin receptor substrate-1/2 (IRS), phosphatidylinositol-3-kinase, (PI3-kinase) and protein kinase C. Although the molecular mechanism is not completely understood, elevated reactive oxygen (ROS) and nitrogen species (RNS) are involved in this process. Elevated ROS/RNS activates nuclear factor-kappaB (NFkB), which promotes the transcription of proinflammatory tumoral necrosis factor alpha (TNFalpha), decreasing the insulin response. Therefore, oxidative stress induced by elevated FA availability may constitute one of the major causes of insulin resistance in skeletal muscle.

Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: indications for a long-chain acyl-CoA ester binding motif

The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K(ATP) channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. Oocytes expressing Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K(ATP) channel and Kir6.2DeltaC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.

Acyl Carrier Protein Synthases from Gram-Negative, Gram-Positive, and Atypical Bacterial Species: Biochemical and Structural Properties and Physiological Implications

Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli, Streptococcus pneumoniae, and Mycoplasma pneumoniae, which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae, like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally, our biochemical studies show that M. pneumoniae AcpS is kinetically a very sluggish enzyme compared with those from E. coli and S. pneumoniae. Together, the results of these studies show that the AcpS enzymes from different bacterial species exhibit different native structures and substrate specificities with regard to the utilization of CoA and its derivatives. These findings suggest that AcpS from different microorganisms plays a different role in cellular physiology.

Acyl Coenzyme A Synthetase Regulation: Putative Role in Long‐Chain Acyl Coenzyme A Partitioning

Long-chain acyl coenzyme A synthetase (ACSL) converts free fatty acids (FFAs) into their metabolizable long-chain acyl coenzyme A (LC-CoA) derivatives that are essential for FFA conversion to CO(2), triglycerides, or complex lipids. ACSL-1 is highly expressed in adipose tissue with broad substrate specificity. We tested the hypothesis that ACSL localization, and resulting local generation of LC-CoA, regulates FFA partitioning. These studies used cell fractionation of rat adipocytes to measure ACSL activity and mass and compared cells from young, mature, fed, fasted, and diabetic rats. Functional studies included measurement of FFA oxidation, complex lipid synthesis, and LC-CoA levels. High ACSL specific activity was expressed in the mitochondria/nuclei (M/N), high-density microsomes (HDM), low-density microsomes (LDM), and plasma membrane (PM) fractions. We show here that, during fasting, total FFA oxidation increased, and, although total ACSL activity decreased, a greater percentage of activity (43 +/- 1.5%) was associated with the M/N fraction than in the fed state (23 +/- 0.3%). In the fed state, more ACSL activity (34 +/- 0.5%) was associated with the HDM than in the fasted state (25 +/- 0.9%), concurrent with increased triglyceride formation from FFA. Insulin increased LC-CoA and ACSL activity associated with the PM. The changes in ACSL activity in response to insulin were associated with only minor changes in mass as determined by Western blotting. It is hypothesized that ACSL plays an important role in targeting FFA to specific metabolic pathways or acylation sites in the cell, thus acting as an important control mechanism in fuel partitioning. Localization of ACSL at the PM may serve to decrease FFA efflux and trap FFA within the cell as LC-CoA.

Minireview: malonyl CoA, AMP-activated protein kinase, and adiposity.

An increasing body of evidence has linked AMP-activated protein kinase (AMPK) and malonyl coenzyme A (CoA) to the regulation of energy balance. Thus, factors that activate AMPK and decrease the concentration of malonyl CoA in peripheral tissues, such as exercise, decrease triglyceride accumulation in the adipocyte and other cells. The data reviewed here suggest that this is related to the fact that these factors concurrently increase fatty acid oxidation, decrease the esterification of fatty acids to form glycerolipids, and, by mechanisms still unknown, increase energy expenditure. Malonyl CoA contributes to these events because it is an allosteric inhibitor of carnitine palmitoyltransferase, the enzyme that controls the transfer of long-chain fatty acyl CoA from the cytosol to the mitochondria, where they are oxidized. AMPK activation in turn increases fatty acid oxidation (by effects on enzymes that govern malonyl CoA synthesis and possibly its degradation) and inhibits triglyceride synthesis. It also increases the expression of uncoupling proteins and the transcriptional regulator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC1alpha), which could possibly increase energy expenditure. Recent studies suggest that the ability of leptin, adiponectin, 5'-aminoimidazole 4-carboxamide riboside (AICAR), adrenergic agonists, and metformin to diminish adiposity may be mediated, at least in part, by AMPK activation in peripheral tissues. In addition, preliminary studies suggest that malonyl CoA and AMPK take part in fuel-sensing and signaling mechanisms in the hypothalamus that could regulate food intake and energy expenditure.

Shared biochemical properties of glucotoxicity and lipotoxicity in islets decrease citrate synthase activity and increase phosphofructokinase activity.

Diabetic states are characterized by a raised serum/islet level of triglycerides and a lowered EC50 (concentration at half-maximal stimulation) for glucose-induced insulin secretion. Culturing islets with long-chain fatty acids (FAs) replicates the basal insulin hypersecretion. In a previous study, we showed that the mechanism involved deinhibition of hexokinase by a 60% decrease in glucose-6-phosphate (G-6-P). The key event was proposed to be an increased phosphofructokinase (PFK) Vmax secondary to an upregulatory effect of the FA metabolite, long-chain acyl-coenzyme A (LC-CoA). We now show another contributory factor, a lowered content of the PFK inhibitor citrate. Citrate synthase Vmax and citrate levels were lowered 45% in rat islets cultured with 250 micromol/l oleate for 24 h. Both effects were reversed by triacsin C, an inhibitor of fatty acyl-CoA synthetase, the enzyme that generates LC-CoA. Culturing islets with high doses of glucose (16.7 mmol/l) for 48 h should also raise cytosolic LC-CoA. As predicted, citrate synthase Vmax was lowered and PFK Vmax was increased, both in a triacsin C-reversible fashion. These results show shared selected functional and biochemical properties in beta-cells of so-called glucotoxicity and lipotoxicity.

MALDI-TOF and electrospray tandem mass spectrometric analysis of fatty acyl-CoA esters

Coenzyme A (CoA) thioesters play a central role in many biochemical reactions, and methods for sensitive identification of these compounds are important for studies of cellular processes. The direct analysis of intact long-chain fatty acyl CoA esters was investigated using matrix assisted laser desorption time-of-flight and electrospray ionization mass spectrometry. Both techniques resulted in the formation of abundant positive and negative ions indicative of the molecular weight of the fatty acyl CoA molecular species. Several structurally significant fragment ions, in particular positive ions indicative of the fatty acyl portion of the molecule, were formed during ionization processes. Metastable (MALDI-TOF) and collision-induced decomposition (quadrupole tandem mass spectrometry) of these molecular ion species resulted in the formation of similar product ions independent of the mode of ionization. Abundant product ions, corresponding to the fatty acyl portion as well as the pantetheine-ADP portion of the molecule, were observed with both ionization techniques. Decomposition of the [M + H] + ion of hexadecyl coenzyme A (C16:0 CoA) at m / z 1006 yielded a prominent product ion at m / z 499 which was characteristic of the fatty acyl portion of the molecule. Similar fatty acyl product ion fragments corresponding to a neutral loss of 609 were found for the [M + H] + species of all the fatty acyl CoA esters investigated. This prominent and diagnostic fragment could be used to detect molecular species of CoA esters present in complex biological mixtures.

Analysis of long-chain fatty acyl coenzyme A. Thioesters by negative ion fast-atom bombardment mass spectrometry and tandem mass spectrometry

Long-chain acyl Coenzyme A (CoA) is essentially composed of three major chemical groups, fatty acyl-, phosphopantetheino-, and 3′,5′,-adenosine diphospho-moieties. The negative ion fast-atom bombardment mass spectrometry spectra of long-chain acyl CoA thioesters were characterized by the formation of abundant [M = H] − and two distinct classes of fragment ions, one class which retained the acyl group and another class which is related to CoA that contains the phosphopantethene and adenine. The ions which retained the acyl group in the spectrum of palmitoyl CoA appeared at m/z 675, 657, 595, and 577 and were found to decompose by loss of alkylketene observed at m/z 357 and 339. Those ions which retained the adenine group were observed at m/z 426 and 408. In contrast to these ions observed following fast-atom bombardment ionization, tandem mass spectrometry of the [M - H] −, from palmitoyl CoA ( m/z 1004), yielded the adenine-containing ions as major products and the acyl-containing ions were of low abundance or not detected. These results suggested that the formation of many characteristic ions observed in direct FAB analysis occurred during the desorption process. The unique relationship between ions which involved the transition from acyl-containing ions to only CoA-containing ions by the loss of alkylketene allowed the development of tandem mass spectrometry protocols for the analysis of acyl CoA mixtures. Precursor scans of either m/z 357 or 339 yielded the identification of each species in a complex mixture. Identification of specific species was obtained with a neutral loss scan of the mass for a specific alkylketene.

Effect of L-Carnitine on Mitochondrial Acyl CoA Esters in the Ischemic Dog Heart

Many studies have shown that L-carnitine has a positive effect on ischemic myocardium, probably by reducing accumulation of long-chain acyl coenzyme A (CoA) esters. Previous studies have involved whole-heart extracts and have not assessed changes of CoA ester levels in mitochondria, the site of translocase inhibition. To more precisely assess L-carnitine effects, we measured long-chain acyl CoA ester levels in cytosol and in mitochondria in the ischemic canine heart. Dogs were divided into four groups: a sham-operated control group; an untreated group; and high- and low-dose L-carnitine-treated groups (30 mg/kg and 100 mg/kg). After 60 min of ischemia, the heart was excised, and the cytosolic and mitochondrial fractions were isolated. CoA esters and the activity of carnitine palmitoylcarnitine transferase (CPT) I and II were measured in both compartments. Approximately 89% of cellular free CoA, 90% of cellular acetyl CoA, 97% of cellular shot-chain acyl CoA, and 92% of cellular long-chain acyl CoA were located in the mitochondrial space under the normal condition. Under the ischemic condition, mitochondrial free CoA was significantly decreased. Conversely, mitochondrial acetyl CoA and long-chain acyl CoA were significantly increased. Treatment with L-carnitine significantly decreased acetyl CoA and long-chain acyl CoA in the ischemic mitochondrial space in a dose-dependent manner. These results support the hypothesis that L-carnitine reduces accumulation of long-chain acyl CoA within the ischemic mitochondrial space and thereby improves mitochondrial function and adenine nucleotide translocation.

Regulation of eicosanoid synthesis in microvessel endothelium: Glucocorticoids do not affect arachidonyl CoA synthetase activity

The properties of acyl coenzyme A (CoA) synthetase activity were characterized in cultured rabbit coronary microvessel endothelial cells. We report here that microvessel endothelial cells contain two long-chain acyl CoA synthetases. One shows activity with a variety of fatty acids, similar to long-chain non-selective fatty acyl CoA synthetases described previously. The other activity was selective for arachidonic acid and other structurally related substrates. Both activities required ATP, Mg 2+ and CoA for optimal activity. The arachidonyl CoA and the non-selective acyl CoA synthetases showed different thermolabilities. Arachidonyl CoA formation was inhibited by greater than 50% after 1 min at 45°C, whereas a 15 min heating treatment was necessary to produce the same relative inhibition of oleoyl CoA synthesis. Glucocorticoid pretreatment (10 −7 M dexamethasone) of the RCME cells did not affect the apparent K m or V max , nor the fatty acid selectivity for either acyl CoA synthetase. Therefore, although fatty acyl CoA synthetases may be involved in limiting eicosanoid formation, these activities do not appear to be glucocorticoid-responsive.

Purification and characterization of fatty acid-binding proteins from human fetal lung.

Fatty acid-binding protein (FABP) was isolated, purified, and characterized from developing human fetal lung cytosol by gel filtration and ion-exchange chromatography. FABP exists in three immunochemically identical forms, DE-I, DE-II, and DE-III, having Mr 15,200 +/- 200 each and isoelectric pH 7.8, 6.9, and 5.4, respectively. DE-I is almost lipid-free, DE-II binds mainly long-chain unsaturated fatty acids, and DE-III is an arachidonic acid carrier. One mole of DE-II and DE-III each binds 1 mol of fatty acids noncovalently. Concentrations of all these FABPs increase gradually from early gestation to term. Defatted lung FABP reverses the inhibitory effect of palmitoyl coenzyme A (CoA) (PAL-CoA) on lung glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the hexose monophosphate (HMP) shunt pathway. This protein when added alone activates the enzyme, suggesting that the original submaximal activity is probably due to the presence of endogenous long-chain fatty acyl CoA esters in the cytosols. As FABP is present in relatively high concentration in most mammalian cells, the potent inhibitory effects of long-chain acyl CoA esters on the HMP shunt pathway in vitro are not seen in intact cells.

Synthesis of docosahexaenoyl-, arachidonoyl- and palmitoyl-coenzyme a in ocular tissues

The synthesis of long-chain acyl coenzyme A (CoA) was studied in the cornea, lens, vitreous, retina and pigment epithelium (PE) in the rat using [ 14C]-labeled palmitic, arachidonic and docosahexaenoic acids as substrates. Except for retina and PE, the ocular tissues studied showed relatively little enzyme activity with the fatty acid substrates. In addition, the enzyme activities were studied in homogenates and microsomal fractions from retina, pigment epithelial cells and choroid of frog, bovine and human eyes. Long-chain acyl CoA synthetase from the microsomal fraction exhibited three- to fivefold greater activity than homogenates in retina and PE. The enzyme activity was highest with palmitic acid, followed by arachidonic acid and docosahexaenoic acid. There were significant differences in enzyme activity between the species. The apparent K m (μ m) and V max [nmol min −1 (mg protein) −1] values for the enzyme in bovine retinal microsomes were 7·91±0·39 ( s.e.) and 21·6±1·04, respectively, for palmitic acid substrate and 5·88±0·25 and 4·58±0·21, respectively, for docosahexaenoic acid substrate. These values for bovine pigment epithelial microsomes were 13·0±0·27 and 36·9±1·18, respectively, for palmitic acid and 15·8±0·40 and 13·2±0·56, respectively, for docosahexaenoic acid. The synthesis of acyl CoA may play a central role in controlling the availability of free arachidonic acid for eicosanoid formation and in the retention of polyunsaturated fatty acid families (18:2, n-6 and 18:3, n-3) within cells of ocular tissues, particularly retina and retinal PE.

Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter.

Long-chain acyl coenzyme A (CoA) synthetase in homogenates and microsomes from rat brain gray and white matter was studied. The formation of the thioesters of CoA was studied upon addition of [1-14C]-labeled fatty acids. The maximal activities were seen with linoleic acid, followed by arachidonic, palmitic, and docosahexaenoic acids in both gray and white matter homogenates and microsomes. The specific activities in microsomes were 3-5 times higher than in homogenates. The presence of Triton X-100 in the assay system enhanced the activity of long-chain acyl CoA synthetase in homogenates. The effect was more pronounced in palmitic and docosahexaenoic acid activation. The apparent Km values and Vmax values for palmitic and docosahexaenoic acids were much lower than for linoleic and arachidonic acids. The presence of Triton X-100 in the medium caused a definite decrease in the apparent Km and Vmax values for all the fatty acids except palmitic acid in which case the reverse was true. There were no significant differences observed in the kinetic measurements between gray and white matter microsomes. These findings are similar to those resulting from the known interference of Triton X-100 in the measurement of kinetic variables of long-chain acyl CoA synthetase of liver microsomes. In this work, no correlation was observed between the fatty acid composition of gray and white matter and the capacity of these tissues for the activation of different fatty acids.

Altered coenzyme A and carnitine metabolism in pressure-overload hypertrophied hearts.

Changes in the total content and acylation state of coenzyme A (CoA) and carnitine in the heart result in alterations in fatty acid metabolism that may be associated with ventricular dysfunction. The present study was undertaken to determine whether abnormal myocardial CoA and carnitine metabolism occur in pressure-overload hypertrophy and congestive heart failure. Right ventricular hypertrophy was induced in cats by pulmonary artery (PA) banding. Total tissue CoA was reduced by 50% in the hypertrophied right ventricle and 25% in the nonhypertrophied left ventricle of PA-banded cats in the presence or absence of heart failure. No alterations were observed in long-chain fatty acyl CoA levels in either ventricle of PA-banded cats. In addition, a 20% reduction in total CoA was also observed in hypertrophied hearts of rats subjected to aortic banding. Although the CoA content was reduced in hypertrophied cat hearts, myocardial tissue levels of its precursor, pantothenic acid, were unaltered. Total tissue carnitine and long-chain fatty acyl-carnitine levels were unchanged in both ventricles of PA-banded cats; however, total carnitine was reduced by 25% in hypertrophied rat hearts. This study establishes a marked reduction in myocardial CoA content in pressure-overload hypertrophy.