Serum ACSL4 levels in patients with ST-segment elevation myocardial infarction (STEMI) and its association with one-year major adverse cardiovascular events (MACE): A prospective cohort study.

In the present prospective cohort research, we aimed to explore the serum levels of Acyl-CoA synthetase long-chain family member 4 (ACSL4) in patients with ST-segment elevation myocardial infarction (STEMI) and its association with 1-year major adverse cardiovascular events (MACE). This prospective cohort study recruited 507 patients who underwent percutaneous coronary intervention for the treatment of STEMI at our hospital during August 2019 to July 2022. The serum ACSL4, tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, and C-reactive protein levels were measured by enzyme-linked immunosorbent assay. Demographic and clinical statistics were also collected. In addition, all patients were followed up for 1 year, and patients with MACE were defined as poor prognosis group. All data used SPSS 26.0 to statistical analyses. The poor prognosis group had significantly higher age and low-density leptin cholesterol (LDLC) levels compared to the favorable prognosis group (P < .05). STEMI patients exhibited significantly elevated serum levels of ACSL4, tumor necrosis factor-α, IL-6, IL-1ß, and C-reactive protein (P < .05). Serum ACSL4 and IL-1ß levels in the poor prognosis group were remarkably enhanced compared to the favorable prognosis group. Curvilinear regression analysis demonstrated that ACSL4 was associated with LDLC and IL-1ß. Moreover, ACSL4 (B = 0.138, 95% CI 1.108-1.189, P < .001), LDLC (B = 2.317, 95% CI 5.253-19.603, P < .001), and IL-1ß (B = 0.061, 95%CI 1.008-1.122, P = .025) levels were the risk factors for STEMI patients with 1-year MACE. This study showed that the serum ACSL4 levels was remarkably elevated in STEMI patients. This study might provide new targets and a comprehensive approach to cardiovascular protection in STEMI patients.

Oxylipin metabolism is controlled by mitochondrial ß-oxidation during bacterial inflammation.

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

ACSL1 affects Triglyceride Levels through the PPARγ Pathway.

In clinical cohort studies, high expression of long-chain acyl-coenzyme A synthetases 1 (ACSL1 gene) in peripheral white blood cells of patients with acute myocardial infarction (AMI) has been utilized as molecular markers of myocardial infarction diagnosis. The plasma triglyceride level of AMI patients is significantly higher than that of healthy individuals. We hypothesized that the high expression of ACSL1 increases the level of triglyceride, which is one of the pathogenesis of AMI promoted by ACSL1. In this report, cell culture based methods were adopted to test the hypothesis and further investigate the effect and mechanism of ACSL1 on lipid metabolism. In this study, liver cells of healthy individuals were cultured, the overexpression and the knockdown vectors of ACSL1 were constructed and transfected into liver cells. The transfection was verified at the mRNA and protein level. Intracellular triglyceride content was quantitatively analyzed using ELISA. Changes of genes related to lipid metabolism were subsequently measured through PCR array. Overexpression of ACSL1 led to higher gene expression and protein levels compared to control and the triglyceride content was significantly increased in overexpressing cells. The expression level of fatty acid oxidation pathway PPARγ was significantly down-regulated compared with the control group, as were genes associated with fatty acid synthesis pathways: SREBP1, ACC, FAS, and SCD1. ACSL1 knockdown decreased the content of triglyceride whereas PPARγ was up-regulated and SREBP1, ACC, FAS, and SCD1 were down-regulated compared with the control group. In summary, high expression of ACSL1 reduced fatty acid ß-oxidation through the PPARγ pathway, thereby increasing triglyceride levels.

X-linked adrenoleukodystrophy in a chimpanzee due to an ABCD1 mutation reported in multiple unrelated humans.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder leading to the accumulation of very long chain fatty acids (VLCFA) due to a mutation in the ABCD1 gene. ABCD1 mutations lead to a variety of phenotypes, including cerebral X-ALD and adrenomyeloneuropathy (AMN) in affected males and 80% of carrier females. There is no definite genotype-phenotype correlation with intrafamilial variability. Cerebral X-ALD typically presents in childhood, but can also present in juveniles and adults. The most affected tissues are the white matter of the brain and adrenal cortex. MRI demonstrates a characteristic imaging appearance in cerebral X-ALD that is used as a diagnostic tool. OBJECTIVES: We aim to correlate a mutation in the ABCD1 gene in a chimpanzee to the human disease X-ALD based on MRI features, neurologic symptoms, and plasma levels of VLCFA. METHODS: Diagnosis of X-ALD made using MRI, blood lipid profiling, and DNA sequencing. RESULTS: An 11-year-old chimpanzee showed remarkably similar features to juvenile onset cerebral X-ALD in humans including demyelination of frontal lobes and corpus callosum on MRI, elevated plasma levels of C24:0 and C26:0, and identification of the c.1661G>A ABCD1 variant. CONCLUSIONS: This case study presents the first reported case of a leukodystrophy in a great ape, and underscores the fidelity of MRI pattern recognition in this disorder across species.

Adult cerebral adrenoleukodystrophy and Addison's disease in a female carrier.

We described a 38-year-old woman of rapidly progressive dementia with white matter encephalopathy and death. She had Addison's disease but the adrenal glands were hyperplastic. Brain magnetic resonance imaging revealed diffuse white matter lesion predominantly in the frontal lobe with band-like contrast enhancement. l-Methyl-11C-methionine positron emission tomography revealed accumulation of tracer in bilateral frontal lobes. Stereotactic biopsy demonstrated demyelination changes. A number of urinary organic acids were elevated. Adrenoleukodystrophy was diagnosed by elevated plasma very long chain fatty acid and ABCD1 gene mutation (C1544C/T). Adrenoleukodystrophy should be considered as a differential diagnosis in women with rapidly progressive white matter encephalopathy.

Influence of breastfeeding on blood-cell transcript-based biomarkers of health in children.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: The expression of specific genes in peripheral blood cells (PBCs) may be used as biomarkers of the metabolic status. High levels of expression of CPT1A, SLC27A2, INSR, LEPR, FASN and PPARα in PBCs are indicative of a lower risk for the insulin resistant or dyslipidaemic state associated with obesity in children. Breastfeeding seems to confer protective effects against obesity and its related metabolic problems. WHAT THIS STUDY ADDS: Children who had been breastfed showed higher expression levels of SLC27A2, FASN, PPARα and INSR in PBCs compared with formula-fed subjects. The relationship of the PBC transcript levels of SLC27A2, INSR, FASN and PPARα with insulin resistance and dyslipidaemia may be dependent on the type of infant feeding (breast vs. formula). The transcript levels of the mentioned biomarkers could be useful to distinguish the formula-fed children who are at higher risk of metabolic alterations. BACKGROUND: Blood-cell transcripts have showed to be good biomarkers of metabolic alterations and their use in early detection and prevention of future disorders is promising. OBJECTIVE: This study aimed to examine the relation between previously proposed transcriptional biomarkers of metabolic health (SLC27A2, CPT1A, FASN, PPARα, INSR, LEPR) in peripheral blood cells and the type of infant feeding in a subset of children from the IDEFICS (Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants) cohort. SUBJECTS: A total of 237 children aged 2-9 years from eight European countries were studied. RESULTS: Breastfed children showed higher expression levels of SLC27A2, FASN, PPARα and INSR, and lower risk of being overweight and of having high plasma triglyceride levels vs. formula-fed children. Besides, overweight formula-fed children presented higher HOMA-index than overweight breastfed children (1.90 vs. 1.62); however, this negative effect was absent in formula-fed children with high expression of SLC27A2. Moreover, formula-fed children with low expression of SLC27A2, FASN, PPARα and INSR presented higher triglyceride levels than subjects with high expression of these genes (77.7 mg dL(-1) vs. 44.8 mg dL(-1) ). This difference was absent in breastfed children. CONCLUSIONS: Protective effects of breastfeeding are reflected in higher expression levels of SLC27A2, FASN, PPARα and INSR in blood cells. These biomarkers may also serve to discriminate the formula-fed children that are at higher risk of metabolic alterations.

Blood cells as a source of transcriptional biomarkers of childhood obesity and its related metabolic alterations: results of the IDEFICS study.

BACKGROUND: IDEFICS (Identification and Prevention of Dietary- and Lifestyle-Induced Health Effects in Children and Infants Project) is a European multicenter study on childhood obesity. One of its goals is to define early biomarkers of risk associated with obesity and its comorbid conditions. OBJECTIVE: We considered blood cells as a new potential source of transcriptional biomarkers for these metabolic disorders and examined whether blood cell mRNA levels of some selected genes (LEPR, INSR, CPT1A, SLC27A2, UCP2, FASN, and PPARα) were altered in overweight children and whether their expression levels could be defined as markers of the insulin-resistant or dyslipidemic state associated with overweight. DESIGN: Blood samples were obtained from 306 normal-weight and overweight children, aged 2-9 yr, from eight different European countries. Whole-blood mRNA levels were assessed by quantitative RT-PCR. RESULTS: LEPR, INSR, and CPT1A mRNA levels were higher in overweight compared with normal-weight children (the two latter only in males), whereas SLC27A2 mRNA levels were lower in overweight children. Significant associations were also found between expression levels of LEPR, INSR, CPT1A, SLC27A2, FASN, PPARα, and different parameters, including body mass index, homeostasis model assessment index, and plasma triglycerides and cholesterol levels. These associations showed that high expression levels of CPT1A, SLC27A2, INSR, FASN, or PPARα may be indicative of a lower risk for the insulin-resistant or dyslipidemic state associated with obesity, whereas low LEPR mRNA levels appear as a marker of high low-density lipoprotein cholesterol, independently of body mass index. CONCLUSIONS: These findings point toward the possibility of using the expression levels of these genes in blood cells as markers of metabolic status and can potentially provide an early warning of a future disorder.

Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo.

Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 +/- 3 vs. 67 +/- 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 +/- 6 vs. 100 +/- 6 nmol/g protein in controls, P < 0.05) and increased [(14)C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 +/- 4 vs. 20 +/- 2 mumol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-(14)C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.

Biochemical effect of intravenous arginine butyrate in X-linked adrenoleukodystrophy.

OBJECTIVES: To determine the biochemical and clinical effects of intravenous arginine butyrate in X-linked Adrenoleukodystrophy (X-ALD). STUDY DESIGN: Arginine butyrate was intravenously infused over a 4-month period in a patient with the rapid cerebral form of X-ALD. Very long chain fatty acids (VLCFA), complete blood counts, and serum chemistries were monitored, and serial MRI of the brain and clinical neurologic examinations were performed. RESULTS: All blood chemical and hematologic values remained within the normal range for age throughout the therapy. After completion of the first day of infusion, the C 26:0 value fell from 1.01 microg/mL to 0.445 microg/mL, which is below the mean value for an X-ALD heterozygote. Throughout the remainder of the trial, all C26:0 levels fell below the mean -1 SD for X-ALD hemizygotes (mean, 1.18 microg/mL, 1 SD = 0.53), ranging from 0.321 to 0.565 microg/mL. Despite reduction of the plasma VLCFA, the patient continued to deteriorate neurologically. CONCLUSIONS: Intravenous arginine butyrate resulted in a rapid decrease in plasma VLCFA but no effect on the neurologic progression of the disease in this patient. Additional studies are needed to determine minimum effective dosage and interval, what proportion of patients respond, and whether the agent can prevent neurologic degeneration.

A third MRX family (MRX68) is the result of mutation in the long chain fatty acid-CoA ligase 4 (FACL4) gene: proposal of a rapid enzymatic assay for screening mentally retarded patients.

BACKGROUND: The gene encoding fatty acid CoA ligase 4 (FACL4) is mutated in families with non-specific X linked mental retardation (MRX) and is responsible for cognitive impairment in the contiguous gene syndrome ATS-MR (Alport syndrome and mental retardation), mapped to Xq22.3. This finding makes this gene a good candidate for other mental retardation disorders mapping in this region. METHODS: We have screened the FACL4 gene in eight families, two MRX and six syndromic X linked mental retardation (MRXS), mapping in a large interval encompassing Xq22.3. RESULTS: We have found a missense mutation in MRX68. The mutation (c.1001C>T in the brain isoform) cosegregates with the disease and changes a highly conserved proline into a leucine (p.P375L) in the first luciferase domain, which markedly reduces the enzymatic activity. Furthermore, all heterozygous females showed completely skewed X inactivation in blood leucocytes, as happens in all reported females with other FACL4 point mutations or deletions. CONCLUSIONS: Since the FACL4 gene is highly expressed in brain, where it encodes a brain specific isoform, and is located in hippocampal and cerebellar neurones, a role for this gene in cognitive processes can be expected. Here we report the third MRX family with a FACL4 mutation and describe the development of a rapid enzymatic assay on peripheral blood that we propose as a sensitive, robust, and efficient diagnostic tool in mentally retarded males.

Identification and molecular characterization of acyl-CoA synthetase in human erythrocytes and erythroid precursors.

Full-length cDNA species encoding two forms of acyl-CoA synthetase from a K-562 human erythroleukaemic cell line were cloned, sequenced and expressed. The first form, named long-chain acyl-CoA synthetase 5 (LACS5), was found to be a novel, unreported, human acyl-CoA synthetase with high similarity to rat brain ACS2 (91% identical). The second form (66% identical with LACS5) was 97% identical with human liver LACS1. The LACS5 gene encodes a highly expressed 2.9 kb mRNA transcript in human haemopoietic stem cells from cord blood, bone marrow, reticulocytes and fetal blood cells derived from fetal liver. An additional 6.3 kb transcript is also found in these erythrocyte precursors; 2.9 and 9.6 kb transcripts of LACS5 are found in human brain, but transcripts are virtually absent from human heart, kidney, liver, lung, pancreas, spleen and skeletal muscle. The 78 kDa expressed LACS5 protein used the long-chain fatty acids palmitic acid, oleic acid and arachidonic acid as substrates. Antibodies directed against LACS5 cross-reacted with erythrocyte membranes. We conclude that early erythrocyte precursors express at least two different forms of acyl-CoA synthetase and that LACS5 is present in mature erythrocyte plasma membranes.

Erythrocyte membrane reacylation in juvenile neuronal ceroid-lipofuscinosis: measurement of membrane-bound carnitine palmitoyl transferase, acyl-CoA synthetase, and lysophospholipid: acyl-CoA acyltransferase activities.

In order to study the biochemical mechanisms responsible for the membrane fatty acid deficiency in juvenile neuronal ceroid-lipofuscinosis, we have analyzed the reacylation pathway in isolated erythrocyte membranes in 5 patients. We studied membrane carnitine palmitoyl transferase, and developed a combined assay to study acyl-CoA synthetase and lysophospholipid acyl-CoA acyltransferase activities. There were no significant differences between control and patient membranes, suggesting that abnormalities in these 3 putative candidate enzymes are not responsible for the disease.

Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates.

1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities. 7. Some of the effects of fibrate treatment can be explained, at least in part, in terms of peroxisomal induction and caution should be exercised in the extrapolation of these results to species, such as man,that are insensitive to peroxisomal proliferation.

Modifications of platelet phospholipid fatty acid composition in streptozocin-induced diabetic rats.

Increased thromboxane A2 (TXA2) generation by platelets has been reported both in diabetic patients and streptozocin-induced diabetic rats. This increase is in contrast to the decreased prostacyclin (PGI2) synthesis by endothelial cells in diabetes. An imbalance in the ratio of TXA2/PGI2 has been implicated in increased platelet aggregation and a high incidence of vascular disease in human diabetes. The mechanism for this imbalance, however, remains elusive. In a previous study from our laboratory, we reported unchanged arachidonic acid levels in platelet membrane phospholipids of 3-week diabetic rats, but a decreased arachidonic acid level in platelet membrane phospholipids of 6-week diabetic rats. In the present communication, we report the role of enzymes that are involved in remodeling arachidonic acid levels of platelet membrane phospholipids in both 3- and 6-week diabetic rats. No alterations were observed in the activities of arachidonoyl-CoA synthetase, acyl-CoA: lysophosphatidylcholine acyltransferase, or phospholipase A2 in platelets from both 3- and 6-week diabetic rats. However, both increased uptake and incorporation of [14C]arachidonic acid into platelets were observed in the diabetic platelet-rich plasma. In conclusion, increased TXA2 formation in diabetic platelets is not due to alterations in the activities of enzymes involved in the incorporation into or release of arachidonate from the diabetic platelet membrane phospholipid, but may be due to increased efficiency of uptake, incorporation or possibly redistribution of this fatty acid among phospholipid classes in diabetic platelets.

Adrenomyeloneuropathy and hypothyroidism. A 15 year follow-up case report.

A case of adrenomyeloneuropathy with diffuse focal demyelination throughout the entire central nervous system, is described in a 29 years old, an adult male, who developed Addison's disease at 14 years of age. Since the age of 23 he has been affected by a progressive spastic paraparesis of the inferior limbs associated with urine incontinence. In the last two years reduced cerebral function, peripheral neuropathy, impotence, primary hypothyroidism and high levels of VLCFA plasma concentration have appeared.

Identity between palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase in human platelet?

Apparent Km values have been determined for the substrates ATP, CoA and fatty acids for the long-chain acyl-CoA synthetase (EC 6.2.1.3) reaction in lysates of human blood platelets. The apparent Km for ATP was higher for saturated fatty acids (C12:0 to C18:0) than for unsaturated acids (C18:1 to C22:6). Other apparent Km values were very similar for all long-chain fatty acids tested. Palmitic acid inhibited the formation of [14C]arachidonoyl-CoA, and arachidonic acid inhibited the formation of [14C]palmitoyl-CoA, with [14C]arachidonate or [14C]palmitate respectively as substrate. After chromatography of Triton X-100-extracted platelet protein in several systems (hydroxyapatite, DEAE-Sepharose, Sephacryl S-200 HR, CoA-Sepharose, Sephadex G-100 and AcA 34), both arachidonoyl-CoA synthetase and palmitoyl-CoA synthetase activities were eluted together in the various protein peaks, and with approximately the same ratio of activities in all peaks. After some purification steps (DEAE-Sepharose and Sephacryl S-200 HR), the acyl-CoA synthetase activity was up to 37 nmol/min per mg of protein with [14C]palmitate as substrate, and up to 116 nmol/min per mg of protein with [14C]arachidonate as substrate. The purification was respectively about 8- and 10-fold. The results indicate that palmitoyl-CoA (or unspecific) synthetase and arachidonoyl-CoA (or specific) synthetase are in fact the same enzyme, in agreement with previously reported results from this laboratory.

Primary stimuli of icosanoid release inhibit arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. Mechanism of action of hydrogen peroxide and methyl mercury in platelets.

Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol.min-1.(10(9) platelets)-1 for the arachidonoyl-CoA synthetase and 37 nmol.min-1.(10(9) platelets)-1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl-CoA synthetase with an IC50 of 3.3 mmol/l without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3-mercapto-1,2-propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl-CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl-CoA synthetase. Methyl mercury left the arachidonoyl-CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 mumol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3-mercapto-1,2-propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+ less than or equal to 1 mmol/l, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol as model stimuli of protein kinase C neither influenced arachidonoyl-CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate-reacylating enzymes arachidonoyl-CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl-CoA synthetase and lysophospholipid acyltransferase.

Identical subcellular distribution of palmitoyl-CoA and arachidonoyl-CoA synthetase activities in human blood platelets.

Fractionation of human blood platelets showed that palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase activities had an identical distribution among subcellular fractions. The activity was highest with arachidonic acid as substrate in all fractions, with an enzyme activity of 50 nmol/min per mg of protein, in a 'dense-tubular-system'-enriched fraction. The ratio activities with arachidonate and palmitate as substrates was about 1.5 in all fractions. Heat inactivation did not distinguish between arachidonoyl-CoA synthetase and a palmitoyl-CoA synthetase. On the other hand, heat inactivation indicated two pools of long-chain acyl-CoA synthetases: one in a mitochondria- and one in the dense-tubular-system-enriched fraction.

Acyl-CoA synthetase activity in Plasmodium knowlesi-infected erythrocytes displays peculiar substrate specificities.

In its blood stages the malaria parasite, Plasmodium, displays very high lipid metabolism. We present evidence for an abundant long-chain acyl-CoA synthetase (EC 6.2.1.3) activity in Plasmodium knowlesi-infected simian erythrocytes. The activity was found to be 20-fold higher in the schizont-infected (the last parasite stage) than in control erythrocytes. The cosubstrate requirements of the enzyme were similar to those previously reported for acyl-CoA synthetases from other sources. Among the separated reaction products of oleyl-CoA synthetase, only PPi and oleyl-CoA were inhibitory, with Ki over 350 microM. The fatty acid specificity of the parasite acyl-CoA synthetase activity was fairly marked and depended on the unsaturation state of the substrate. The tested fatty acids displayed similar Vmax, whereas their Km ranged from 11 (palmitate) to 59 microM (arachidonate). Finally, experiments involving heat inactivation and separation on hydroxyapatite excluded the presence of a specific arachidonyl-CoA synthetase identical to those present in other cells. On the other hand, fatty acid competition experiments evidenced the existence of at least two distinct enzymatic sites for fatty acid activation in P. knowlesi-infected simian erythrocytes: one is specific for saturated fatty acids and the other for polyunsaturated species, whereas oleate could be activated at both sites.

Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid.

The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins. This pathway has been characterized further by studying the activation and incorporation of [9,10(n)-3H]oleic acid and transesterification of [1-14C]oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts. Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars. Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid. Compared to ghosts, the ability of inside-out vesicles to activate and incorporate [9,10(n)-3H]oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify [1-14C]oleoyl-CoA to phospholipid is diminished by over 50%. These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities. This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation.