Glucosylceramide synthase inhibition with lucerastat lowers globotriaosylceramide and lysosome staining in cultured fibroblasts from Fabry patients with different mutation types.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene coding for α-galactosidase A (α-GalA). The deleterious mutations lead to accumulation of α-GalA substrates, including globotriaosylceramide (Gb3) and globotriaosylsphingosine. Progressive glycolipid storage results in cellular dysfunction, leading to organ damage and clinical disease, i.e. neuropathic pain, impaired renal function and cardiomyopathy. Many Fabry patients are treated by bi-weekly intravenous infusions of replacement enzyme. While the only available oral therapy is an α-GalA chaperone, which is indicated for a limited number of patients with specific 'amenable' mutations. Lucerastat is an orally bioavailable inhibitor of glucosylceramide synthase (GCS) that is in late stage clinical development for Fabry disease. Here we investigated the ability of lucerastat to lower Gb3, globotriaosylsphingosine and lysosomal staining in cultured fibroblasts from 15 different Fabry patients. Patients' cells included 13 different pathogenic variants, with 13 cell lines harboring GLA mutations associated with the classic disease phenotype. Lucerastat dose dependently reduced Gb3 in all cell lines. For 13 cell lines the Gb3 data could be fit to an IC50 curve, giving a median IC50 [interquartile range (IQR)] = 11 µM (8.2-18); the median percent reduction (IQR) in Gb3 was 77% (70-83). Lucerastat treatment also dose dependently reduced LysoTracker Red staining of acidic compartments. Lucerastat's effects in the cell lines were compared to those with current treatments-agalsidase alfa and migalastat. Consequently, the GCS inhibitor lucerastat provides a viable mechanism to reduce Gb3 accumulation and lysosome volume, suitable for all Fabry patients regardless of genotype.

Lucerastat, an iminosugar with potential as substrate reduction therapy for glycolipid storage disorders: safety, tolerability, and pharmacokinetics in healthy subjects.

BACKGROUND: Lucerastat, an inhibitor of glucosylceramide synthase, has the potential to restore the balance between synthesis and degradation of glycosphingolipids in glycolipid storage disorders such as Gaucher disease and Fabry disease. The safety, tolerability, and pharmacokinetics of oral lucerastat were evaluated in two separate randomized, double-blind, placebo-controlled, single- and multiple-ascending dose studies (SAD and MAD, respectively) in healthy male subjects. METHODS: In the SAD study, 31 subjects received placebo or a single oral dose of 100, 300, 500, or 1000 mg lucerastat. Eight additional subjects received two doses of 1000 mg lucerastat or placebo separated by 12 h. In the MAD study, 37 subjects received placebo or 200, 500, or 1000 mg b.i.d. lucerastat for 7 consecutive days. Six subjects in the 500 mg cohort received lucerastat in both absence and presence of food. RESULTS: In the SAD study, 15 adverse events (AEs) were reported in ten subjects. Eighteen AEs were reported in 15 subjects in the MAD study, in which the 500 mg dose cohort was repeated because of elevated alanine aminotransferase (ALT) values in 4 subjects, not observed in other dose cohorts. No severe or serious AE was observed. No clinically relevant abnormalities regarding vital signs and 12-lead electrocardiograms were observed. Lucerastat Cmax values were comparable between studies, with geometric mean Cmax 10.5 (95% CI: 7.5, 14.7) and 11.1 (95% CI: 8.7, 14.2) µg/mL in the SAD and MAD study, respectively, after 1000 mg lucerastat b.i.d. tmax (0.5 - 4 h) and t1/2 (3.6 - 8.1 h) were also within the same range across dose groups in both studies. Using the Gough power model, dose proportionality was confirmed in the SAD study for Cmax and AUC0-∞, and for AUC0-12 in the MAD study. Fed-to-fasted geometric mean ratio for AUC0-12 was 0.93 (90% CI: 0.80, 1.07) and tmax was the same with or without food, indicating no food effect. CONCLUSIONS: Incidence of drug-related AEs did not increase with dose. No serious AEs were reported for any subject. Overall, lucerastat was well tolerated. These results warrant further investigation of substrate reduction therapy with lucerastat in patients with glycolipid storage disorders. SAD study was registered on clinicaltrials.gov under the identifier NCT02944487 on the 24th of October 2016 (retrospectively registered). MAD study was registered on clinicaltrials.gov under the identifier NCT02944474 on the 25th of October 2016 (retrospectively registered). TRIAL REGISTRATION: A Study to Assess the Safety and Tolerability of Lucerastat in Subjects With Fabry Disease. Clinicaltrials.gov: NCT02930655 .

Effect of albumin, low temperature and metabolic inhibitors on transport of fatty acids into cultured human leukemic myeloid cells.

Radioactively-labelled palmitic acid was used to study the effects of albumin, low temperature and several inhibitors of metabolism on transport of fatty acids into cultured human leukemic myeloid cells. When serum or albumin were present in the medium, uptake of fatty acid by cells as well as its further incorporation into phospholipids and neutral lipids were considerably reduced. Uptake and metabolic utilization of this fatty acid was reduced at low temperature, in the presence or absence of albumin in the incubation medium. In absence of albumin, addition of iodoacetate, sodium cyanide or sodium azide had but little effect on the total uptake of fatty acids while metabolic utilization was reduced. When albumin was present, these inhibitors reduced both total uptake and incorporation into lipids. The data suggest that incorporation of the fatty acid into the outer layer of the cell membrane is controlled by the concentration of free, uncomplexed molecules of fatty acid adjacent to the cell surface. In the absence of albumin this is a fast reaction which reaches nearly maximal uptake in three minutes. In the presence of albumin, this process is much slower and follows a nearly linear course between 3 and 60 minutes. Translocation into the inner layer of the membrane and subsequent utilization for metabolic processes is a much slower process, which seems to depend on the quantity of the fatty acid in the outer layer.

Transport of fatty acids across the membrane of human erythrocyte ghosts.

Human erythrocyte ghosts were used for studying the mechanism of uptake and membrane transport of fatty acids. Hemoglobin-free ghosts were prepared and loaded with substrates such as CoA and/or ATP, and their ability for transporting and activating radiolabelled palmitic acid was tested further. Uptake of radiolabelled palmitic acid by CoA- and ATP-loaded ghosts exceeded that observed with ghosts loaded only with ATP, the latter being greater than that measured with non-loaded ghosts. Acyl-CoA was synthesized in CoA- and ATP-loaded ghosts upon incubation with radiolabelled palmitic acid. Both CoA and ATP were needed within the ghosts to permit acyl-CoA synthesis, suggesting that the acyl-CoA synthetase is located in and is bound to the inner layer of the membrane. The rate of acyl-CoA synthesis was saturable with increasing concentration of palmitic acid in the incubation mixture, and kinetic parameters were calculated. The rate of acyl-CoA synthesis in CoA- and ATP-loaded ghosts upon incubation with radiolabelled palmitic acid was markedly decreased when increasing albumin concentration in the incubation medium up to a molar ratio albumin/fatty acid of one to one. It is not easy to distinguish experimentally fatty acids located in the outer layer and the inner layer of the membrane and the data of this paper suggest that acyl-CoA synthesis by an enzyme located in the inner layer could be used as a measure of the acyl groups which have been translocated across the membrane of erythrocyte ghosts.

Induction of lipid storage in cultured leukemic myeloid cells by pyrene-dodecanoic acid.

When incubated for 1-3 days in the presence of the fatty acid analog, 12-(1-pyrene)dodecanoic acid, the neutral lipid content of cultured human leukemic myeloid cells increased considerably, while that of the phospholipids increased to a much lesser extent. Among the neutral lipids, di- and monoacylglycerols predominated and a considerable portion of the fatty acyl residues of these newly synthesized neutral lipids consisted of pyrene-dodecanoic acid. Light microscopy showed evidence for the presence of highly fluorescent lipid droplets within the cells. Electron microscopy showed lipid globules, mostly devoid of a unit membrane, multivesicular inclusion bodies and some multilamellar membranous structures. In comparison, cells incubated with palmitic acid show neither these cellular structures, nor the increase of the neutral lipid content. The lipid storage, induced by pyrene-dodecanoic acid, is probably related to ineffective degradation of this fatty acid analog and might serve as an experimental model of cellular lipidosis.

Transport of fluorescent derivatives of fatty acids into cultured human leukemic myeloid cells and their subsequent metabolic utilization.

Transport of fluorescent derivatives of fatty acids across the cell membrane of cultured human leukemic myeloid cells (HL 60) and their subsequent metabolic utilization were studied. The rates of uptake of these derivatives and their incorporation into cellular lipids wer compared with that of radioactively labelled palmitic acid. Three groups of fluorescent derivatives were observed: A, those transported into the cells and subsequently incorporated into neutral lipids and phospholipids, B, fatty acids which were taken up by the cells but not utilized metabolically, and C, fatty acids which were not transported across the cell membrane. Fatty acids of the latter group, except the hydrophobic probe, also contained functional groups such as hydroxy, acetylamino or sulfonylamino. When observed in fluorescence microscopy, cells incubated with group A fatty acids contained intracellular fluorescent granules, whereas those incubated with group B fatty acids showed diffuse fluorescence. HL 60 cells undergo differentiation into granulocytes or macrophages upon treatment with dimethylsulfoxide or a phorbol ester, respectively. When compared to the uninduced cells, the transport of the fluorescent fatty acids or palmitic acid as well as their subsequent incorporation into lipids were considerably lower in the granulocytes and higher in the macrophages. The use of the fluorescent derivatives as a tool for studying transport of fatty acids across the cell membrane is discussed.

A rapid selection for animal cell mutants with defective peroxisomes.

Chinese hamster ovary (CHO) cells take up and incorporate 9-(1'-pyrene)nonanol (P9OH) into phospholipids and neutral lipids. Exposure of P9OH-labeled cells to long wavelength ultraviolet (UV) light causes cell death, because excitation of the pyrene moiety generates reactive oxygen species. CHO mutant cells deficient in plasmalogen biosynthesis and peroxisome assembly (Zoeller, R.A. and Raetz, C.R.H. (1986) Proc. Natl. Acad. Sci. USA 83, 5170-5174) are much more resistant to P9OH/UV treatment than are wild-type cells. This phenotype is explained by a 7.5-fold reduction of P9OH incorporation into the ethanolamine-linked phospholipids in the mutant cells and 2.4- to 6-fold reduction of P9OH incorporation into all other phospholipids and triglycerides, suggesting a general defect in fatty alcohol metabolism. [U-14C]Hexadecanol incorporation into the phospholipids of the mutant cells is also impaired. In contrast, the fatty acid analog, 9-(1'-pyrene)nonanoic acid, is incorporated into cells two times more rapidly by the mutants than by the wild type. Resistance to P9OH/UV treatment affords a simple, new method for the selection of animal cell mutants defective in peroxisome biogenesis.

Effects of chronic aortic coarctation on atherosclerosis and arterial lipid accumulation in the Watanabe hereditary hyperlipidemic (WHHL) rabbit.

The effects of high blood pressure on atherosclerosis were examined in the Watanabe heritable hyperlipidemic (WHHL) rabbit. For this purpose, the subdiaphragmatic aorta of rabbits was partially ligated (coarctation) to increase blood pressure. Atherosclerosis was assessed 4 months later by morphometric analyses and quantitation of arterial lipids. Results were compared to control WHHL rabbits with matched plasma triglycerides and cholesterol levels. A marked increase in atherosclerotic lesions was observed in the thoracic aorta of the hypertensive rabbits without qualitative changes in its morphometric features. The cross sectional area of the atherosclerotic plaques of the ascending and descending aorta in the hypertensive rabbits was two- and six-times larger than in normotensive rabbits, respectively. Lesions represented 12.0% +/- 3.5% of the total medial cross sectional area of the descending aorta of normotensive rabbits, versus 45.0% +/- 5.7% in hypertensive rabbits. No lesions were observed downstream of the coarctation in hypertensive rabbits, nor in the normotensive rabbits. Accumulation of cholesterol and choline-containing phospholipids in the descending aorta of hypertensive rabbits was increased 3.2- and 1.5-fold, respectively, when compared to normotensive rabbits. Hypertension did not change the unesterified cholesterol/total cholesterol and sphingomyelin/lecithin + lysolecithin molar ratios. In conclusion, chronic coarctation enhances the atherosclerotic response in WHHL rabbits in the high blood pressure compartment, and reduces the variability of this response.

Down-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels and synthesis in syrian hamster C100 cells by the oxidosqualene cyclase inhibitor [4'-(6-allyl-ethyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromophenyl)-me thanone (Ro 48-8071): comparison to simvastatin.

In vivo inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC, E.C. 5.4.99.7)--the enzyme which catalyzes the cyclization of monooxidosqualene to lanosterol--does not result in elevated 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) activity. This trait is attributed to increased levels of oxysterols, produced upon partial inhibition of OSC, that suppress HMGR and other sterol-responsive genes. The OSC inhibitor [4'-(6-allyl-ethyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromopheny l)-methanone (Ro 48-8071) was shown earlier to lower low-density lipoprotein (LDL) cholesterol in hamsters with no increase in hepatic HMGR, in contrast to simvastatin. To delineate the regulatory mechanism(s) by which Ro 48-8071 reduces cholesterol synthesis without raising HMGR levels, Syrian hamster C100 cells were incubated with either Ro 48-8071 or simvastatin, and their effects on cholesterol synthesis and LDL uptake, as well as on HMGR mRNA levels and rates of synthesis, were determined. Using RNase protection and radioimmunoprecipitation assays, we found that, in the absence of LDL in the culture medium, both HMGR mRNA levels and synthesis were reduced with concentrations of Ro 48-8071 inhibiting cholesterol synthesis by 50-75%, whereas LDL uptake was either reduced or unchanged. In contrast, simvastatin, at concentrations inhibiting cholesterol synthesis by the same 50-75%, increased both HMGR mRNA levels and synthesis, as well as LDL uptake. In the presence of LDL, HMGR mRNA levels and synthesis along with LDL uptake were little affected after incubation with Ro 48-8071. Still, simvastatin markedly increased both HMGR mRNA levels and synthesis in cells incubated in the presence of LDL, leaving LDL uptake unaffected. These data suggest that inhibition of OSC by Ro 48-8071 results in an indirect down-regulation of HMGR mRNA levels and synthesis.

Exogenous [1-(14)C]lignoceric acid uptake by neurons, astrocytes and myelin, as compared to incorporation of [1-(14)C]palmitic and stearic acids.

In order to compare the incorporation of several saturated fatty acids into the brain, radioactive palmitic, stearic and lignoceric acids were injected into mice. The radioactivity was measured in lipids from isolated neurons, astrocytes and myelin. Our data indicate that specific radioactivity of lignoceric acid after its injection was very high in neurons and astrocytes when comparing with serum lignoceric acid specific radioactivity: evidence of the uptake of exogenous lignoceric acid by brain cells and myelin is provided. The incorporation of exogenous palmitic acid into brain cells was much higher than the incorporation of exogenous stearic acid. We hypothesize that exogenous saturated fatty acid uptake is selective in relation with the acyl chain length and the intracerebral synthesis.

In vivo incorporation of exogenous [1-14C]stearic acid into neurons and astrocytes.

Exogenous stearic acid is needed to synthesize the membranes of neurons and astrocytes. Subcutaneously injected [1-14C]acid is taken up through the 'blood brain barrier' and incorporated into lipids of both cell types, the specific radioactivity being higher in astrocytes as compared to neurons (2200 and 800 cpm/mg proteins, respectively), 20 h after injection. Phospholipids contain high amount of radioactivity (80% in astrocytes, 65% in neurons); glycosphingolipids contain low quantities of label in the two cell types. The injected acid is partly metabolized in the brain by elongation and desaturation (thus providing very long chains, saturated mono-unsaturated and poly-unsaturated); it is also partly degraded into acetate units (utilized for synthesis of palmitic acid).

The pharmacokinetics and tissue distribution of the glucosylceramide synthase inhibitor miglustat in the rat.

Miglustat (Zavesca) is a reversible inhibitor of glucosylceramide synthase, which catalyses the first step in the glucosylceramide biosynthetic pathway, and is approved for therapy in patients with type 1 Gaucher disease. The present report describes the pharmacokinetic profile of miglustat in the rat with a focus on tissue distribution. Experiments were performed with radiolabeled miglustat itself and with a perbutyrated prodrug, the latter being readily converted to miglustat during gastrointestinal absorption and first pass metabolism. Miglustat was well absorbed and exhibited an oral bioavailability of 40-60%. Tissue distribution studies indicated the presence of miglustat in a number of organs and tissues that are considered of importance for the long-term therapeutic benefit, in particular the central nervous system, bone and lung. Miglustat was eliminated via renal clearance by a combination of glomerular filtration and active secretion. Hepatic clearance was negligible, as was the role of metabolism in the overall elimination process of miglustat in the rat.

Photosensitization to ultraviolet irradiation and selective killing of cells following uptake of pyrene-containing fatty acid.

Cells were incubated with 12-(1-pyrene)-dodecanoic acid (P12), a long-chain fatty acid to which a pyrene ring has been attached covalently. This acid was transported across the plasma membranes of cells and subsequently incorporated into their neutral lipids and phospholipids. Irradiation of these pyrene-containing cells for short periods (0.5-4 min) with ultraviolet light at 366 nm resulted in eventual cell death. Similar irradiation had no effect on cells that had not been exposed to P12. The time of the period of irradiation necessary for inducing the toxic process was related to the quantity of P12 incorporated, the latter being a function of the respective metabolic activity of the individual cell type. The degree of incorporation of P12 into a cell, and consequently its acquired sensitivity to killing by ultraviolet irradiation at 366 nm, was affected by the incubation temperature and addition of non-fluorescent fatty acid, albumin or other serum proteins. Different degrees of incorporation of P12 into various cell types were used for selective killing and elimination of cell populations by irradiation at 366 nm. The combined procedure of preincubation with P12 followed by ultraviolet irradiation thus permitted selection of cell types with a greater resistance to this procedure.

Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation.

Chinese hamster ovary cells (CHO-K1) photosensitized with 12-(1'-pyrene)dodecanoic acid (P12) are killed when exposed to long wavelength ultraviolet (UV) light (greater than 300 nm). Mutants deficient in plasmalogen biosynthesis are hypersensitive to this treatment. We now demonstrate that plasmenylethanolamine is rapidly and preferentially destroyed when CHO-K1 cells, photosensitized either with P12 or merocyanine 540, are irradiated with light of the appropriate wavelength. Using [2-14C]ethanolamine, [1-14C]hexadecanol, or [U-14C]hexadecanol to follow the turnover of plasmenylethanolamine, we show that 2-monoacylglycerophosphoethanolamine, formic acid, and pentadecanal are formed during P12/UV treatment of CHO-K1 cells, but not of mutant cells deficient in plasmalogen synthesis. The decomposition of plasmenylethanolamine is O2-dependent, is enhanced in D2O, and is reduced in the presence of sodium azide. The process may be explained, in part, by the cycloaddition of singlet oxygen to the vinyl ether linkage of plasmenylethanolamine, generating a dioxetane intermediate that would be expected to decompose under physiological conditions to the observed products. An additional possibility is the formation of an allylic hydroperoxide at the 1'-carbon of the alkyl moiety by an "ene" reaction of singlet oxygen, or by radical-mediated oxidation, followed by metabolism or chemical decomposition of the hydroperoxide. Given the P12/UV hypersensitivity of plasmalogen-deficient mutants, we suggest that plasmalogens might protect animal cell membranes from singlet oxygen and/or radical-initiated oxidation by functioning as scavengers and decomposing to products that can be reutilized.

A possible role for plasmalogens in protecting animal cells against photosensitized killing.

Chinese hamster ovary (CHO) cells incorporate 12-(1'-pyrene) dodecanoic acid (P12) into membrane lipids. Exposure of P12-labeled cells to long wavelength ultraviolet light causes cell killing, presumably because excitation of the pyrene moiety (a photosensitizer) leads to the generation of reactive oxygen species. Cytotoxicity is dependent upon the concentration of P12 used to label the cells, and time of UV exposure, and the presence of oxygen during irradiation. CHO mutant cells deficient in plasmalogen biosynthesis and peroxisome assembly (Zoeller, R.A., and Raetz, C.R.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170-5174) are several orders of magnitude more sensitive to P12/UV treatment than wild-type cells, permitting direct selection of one wild-type cell in 1 X 10(4) mutant cells. A major factor responsible for the P12/UV hypersensitivity of these mutants appears to be the absence of plasmalogens. Supplementation of the mutants with 1-O-hexadecyl-sn-glycerol restores plasmalogen levels and nearly normal resistance to P12/UV treatment, whereas the biogenesis of peroxisomes is not restored. The P12/UV hypersensitivity of the plasmalogen-deficient mutants, together with the selective, P12/UV-induced decomposition of plasmalogens in wild-type cells, documented in the accompanying manuscript, suggest that the vinyl ether linkage of plasmalogens plays a direct role in protecting animal cell membranes against certain oxidative stresses.

Cytoplasmic requirement for peroxisome biogenesis in Chinese hamster ovary cells.

Hybrids constructed by fusion of wild-type Chinese hamster ovary cells (CHO-K1) to peroxisome-deficient CHO mutants (ZR-78.1) contain normal peroxisomes, demonstrating that the mutation(s) are recessive. "Nuclear hybrids" prepared by fusion of CHO-K1 karyoplasts to mutant ZR-78.1 occasionally fail to regain intact peroxisomes (approximately 1/300 cells). These peroxisome-deficient nuclear hybrids closely resemble the original mutant cells by biochemical criteria, but their modal chromosome number is 36-38, the same as that of CHO hybrids generated from intact cells. When the peroxisome-deficient nuclear hybrids are fused to wild-type cytoplasts, a fraction of the fusion products (at least 70%) continue to propagate normal peroxisomes indefinitely. Peroxisome biogenesis cannot be reinitiated in cells of mutant ZR-78.1 by fusion to wild-type cytoplasts. Our results suggest that a wild-type nucleus by itself is necessary but not sufficient for restoration of normal peroxisome biogenesis and that a cytoplasmic component of wild-type cells, possibly a normal peroxisome, is also required.

Purified rat brain microvessels exhibit both acid and neutral sphingomyelinase activities.

Purified rat brain microvessels have been shown to hydrolyze radiolabeled sphingomyelin by means of two different enzyme systems. Enzymatic activity was detected at pH 7.4 and was strongly stimulated by magnesium or manganese and inhibited by calcium. Activity at pH 5.1 could also be found and was not dependent on any of these cations. At neutral pH and in the presence of magnesium, the rate of sphingomyelin hydrolysis did not exhibit a linear relationship with protein concentration. In contrast, increasing the protein concentration from 0.05 to 0.5 mg/ml resulted in a constant increase of sphingomyelin hydrolysis at pH 5.1. Kinetic parameters of both neutral and acid activities have been determined and were similar in magnitude to values reported previously for neural sphingomyelinases. This work demonstrates the occurrence of a neutral sphingomyelinase activity in purified rat brain microvessels, an observation raising the question of its role at the level of the blood-brain interface.

Arachidonoyl-coenzyme A synthetase and nonspecific acyl-coenzyme A synthetase activities in purified rat brain microvessels.

Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)