Short term regulation of acyl CoA: Cholesterol acyl transferase (ACAT) activity in the regenerating and perinatal liver

Acyl coenzyme A:cholesterol acyltransferase (ACAT), the enzyme catalyzing the hepatic cholesterol esterification could be involved in the modified availability of cholesterol detectable in proliferating systems. While no significant variations are detectable in the regenerating liver, the modified ACAT activity during liver development and its differential sensitivity to the in vitro stimulation of modulatory systems suggest an involvement of the enzyme in this proliferating process.     

Regulation of acyl CoA: Cholesterol acyl transferase (ACAT) activity by mevalonate and cholesterol in isolated rat hepatocytes during perinatal development

Acyl CoA: cholesterol acyl transferase (ACAT) activity presents marked oscillations and differential sensitivity to the in vitro stimulation of the kinase-phosphatase modulatory system in the perinatal rat liver.The regulation of this enzyme activity by some modulators generally active in adulthood, such as cholesterol, lipoproteins and mevalonate, has been studied in hepatocytes isolated at different developmental stages. A lack of effect of mevalonate and a positive effort of lipoprotein cholesterol have been observed at the fetal and neonatal stages.A differential prevalence is suggested of one of the two modulatory mechanisms (phosphorylation-dephosphorylation system, or substrate effect) at each developmental stage.     

Acyl transferase activities in dog lung microsomes

Mammalian lung has a high concentration of dipalmitoyl phosphatidylcholine and other phospholipids in which both fatty acid ester chains are saturated, as opposed to the usual asymmetric phospholipid (one saturated fatty acid and one unsaturated fatty acid). The acyl transferase system in dog lung microsomes was studied by determining the reactivities of various acyl CoA derivatives with 1-lyso-2-acyl- and 1-acyl-2-lyso-phosphatidylcholine. The 16:0 derivative had equal reactivity for both the 1- and 2-lyso positions. The 18:0 derivative also exhibited marked reactivity toward both positions, although the specific activity of the enzyme when palmitoyl CoA was used was approximately twice that compared to when stearoyl CoA was used. The 16:1 derivative showed approximately the same reactivity toward the 1-lyso position as did 16:0 but both 16:1 and 18:1 were more active with the 2-lyso position. These results suggest that acyl transferases may be important in the lung to insure that sufficient amounts of dipalmitoyl phosphatidylcholine will always be present for use in pulmonary surfactant biosynthesis. It is also conceivable that the acyl transferase system described acts on 1- and 2-lyso-palmitoyl phosphatidylcholine (produced by phospholipase hydrolysis of dipalmitoyl phosphatidylcholine) in order to produce phosphatidylcholine species needed for cellular purposes other than surfactant function.     

Accessibility of lysolecithin in catecholamine secretory vesicles to acyl CoA:lysolecithin acyl transferase

Lysolecithin (monoacylglycerophosphorylcholine) accounts for 13 to 20% of the lipid phosphorous of the bovine adrenal catecholamine secretory vesicles (chromaffin granules). We have incubated purified vesicles with [1-¹⁴C] oleyl coenzyme A and rat liver microsomes containing acyl coenzyme A: monoacylglycerophosphorylcholine acyl transferase to determine the accessibility of the granule membrane lysolecithin to another membrane. No acylation of lysolecithin occurs when the chromaffin granules are intact. The accessibility of the granule membrane lysolecithin increases markedly when the vesicles are broken.     

The subcellular distribution of acyl CoA: Dihydroxyacetone phosphate acyl transferase in guinea pig liver

Upon differential centrifugation, the enzyme acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42) in guinea pig liver is shown to sediment in a lysosomal-peroxisomal fraction. Comparison of the distribution of the marker enzymes and of DHAP acyl transferase indicates that the acyl transferase is localized in peroxisomes (microbodies).     

Erythrocyte membrane acyl:CoA synthetase activity

The presence of long-chain acyl:CoA synthetases in mammalian microsomes and mitochondria has been established previously [(1971) Biochim. Biophys. Acta 231, 32-47]. The presence of a plasma membrane-associated enzyme was investigated in human erythrocyte ghost plasma membranes, where an enzyme exhibiting high activity, and with a preferred substrate of 18 carbon chain length, was discovered. The results are consistent with the presence of a single enzyme. The effect of the degree of unsaturation of the fatty acid substrates was not as pronounced as that arising from the length of the carbon chain. The pattern of substrate preference of the enzyme was omega 3 polyenoics greater than omega 6 polyenoics greater than omega 9 monoenoics greater than saturated fatty acids. This may relate to the similar substrate preference pattern exhibited by the fatty acyl desaturase enzymes. However, the role played by long-chain acyl:CoA synthetase in erythrocyte metabolism is uncertain, but may relate to the transportation of polyenoic fatty acids in the circulation.     

Lysophosphatidylserine dependent incorporation of acyl CoA into phospholipids in rat brain microsomes

[¹⁴C]OleoylCoA was incorporated into phosphatidylinositol 4$\text{1}\text{2}$ times more efficiently than into phosphatidylserine in rat brain and liver microsomes when incubated with various levels of 1-acyl-sn-glycero-3-phosphoserine. In contrast, 1-acyl-sn-glycero-3-phosphocholine dependent incorporation of oleoylCoA was only into phosphatidylcholine. When [l-³H]serine labeled 1-acyl-sn-glycero-3-phosphoserine was used as the labeled substrate, no phosphatidylserine synthesis could be detected in rat brain microsomes. OleoylCoA incorporation in phospholipids in the presence of lysophosphatidylserine was primarily at the 2-position while stearoylCoA was incorporated at the 1-position. These results are interpreted to suggest that there is no acylCoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase in rat brain microsomes and the lysophosphatidylserine dependent position-specific incorporation of acylCoA into various phospholipids may be due to an exchange reaction. A simple highly reproducible one dimensional thin-layer chromatographic system is described for the separation of all the major phospholipids of brain and liver.     

Inhibition of human placental progesterone and estrogen synthesis in early human gestation by aminoglutethimide in vivo

The inhibitory effect of d,l-aminoglutethimide (AG) on the synthesis of progesterone and estradiol in early human pregnancy (8th–12th week of gestation) was investigated in volunteers; control group (n = 11), AG group [1000 mg AG orally at test begin (n = 6)]. Venous blood samples were taken at the beginning of the test and 0.5, 1, 2, 4, 8 and 24 h thereafter. In controls, no significant changes in serum progesterone and estradiol could be observed during 24 h. In the AG group, a decrease in progesterone and estradiol could be observed within 1 h after the test began; lowest serum steroid concentrations were reached after 4 h. Relative to the initial values taken as 100%, the greatest decrease in progesterone ranged between 37 and 83%, 62 15% (x SD) (n = 6); the greatest decrease in estradiol ranged between 32 and 78%, 51 17% (x SD) (n = 6). Twenty four hours after AG treatment, both steroids reached similar concentrations to those found at test begin. No clinical signs (e.g. uterine bleeding, contractions) for the abortifacient action of AG were observed.In conclusion, a single dose of AG (1000mg given orally) cannot induce a therapeutic abortion in early pregnancy. In accordance with in vitro studies, the inhibitory effect of AG on placental progesterone formation is due to an inhibition of mitochondrial cholesterol side chain cleavage. The decrease in estradiol is thought to be related to an inhibition of placental aromatase.     

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 apparentK _m values andV _max 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 apparentK _m and Vmax values for all the fatty acid 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.     

NADPH- and iron-dependent lipid peroxidation inhibit aromatase activity in human placental microsomes

During pregnancy placenta is the most significant source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides and other ROS is often linked to pre-eclampsia. It is already proved that placental endoplasmic reticulum may be an important place of lipid peroxides and superoxide radical production. In the present study we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) inhibit placental aromatase--a key enzyme of estrogen biosynthesis in human placenta. We showed that significant inhibition of this enzyme is caused by small lipid peroxidation (TBARS (thiobarbituric acid-reactive substances)<4nmol/mg microsomal protein (m.p.)). More intensive lipid peroxidation (TBARS>9nmol/mg microsomal protein) diminishes aromatase activity to value being less than 5% of initial value. NADPH- and iron-dependent lipid peroxidation also causes disappearance of cytochrome P450 parallel to observed aromatase activity inhibition. EDTA, alpha-tocopherol, MgCl(2) and superoxide dismutase (SOD) prevent aromatase activity inhibition and cytochrome P450(AROM) degradation. Mannitol and catalase have not effect on TBARS synthesis, aromatase activity and cytochrome P450 degradation. In view of the above we postulate that the inhibition of aromatase activity observed is mainly a consequence of cytochrome P450(AROM) degradation induced by lipid radicals. The role of hydroxyl radical in cytochrome P450 degradation is negligible in our experimental conditions. The results presented here also suggest that the inhibition of aromatase activity can also take place in placenta at in vivo conditions.     

A novel lysophosphatidylcholine acyl transferase activity is expressed by peroxiredoxin 6

The phospholipase A₂ (PLA₂) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation were K_m 18 μM and V_max 30 nmol/min/mg protein; the V_max was increased 25-fold by phosphorylation of the protein while K_m was unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA₂ activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA₂ and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.     

The constitutive 7-ethoxycoumarin O-deethylase activity of human placental microsomes: relationship to aromatase

The constitutive 7-ethoxycoumarin deethylase activity of human placental microsomes from non-smokers was acutely inhibited by a number of androgens which serve as substrates for and/or competitive inhibitors of estrogen synthesis by the aromatase activity of these preparations. 10 beta-(2-Propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione, androgen derivatives which produce a mechanism-based, time-dependent inactivation of placental aromatase caused a cofactor-dependent decay in deethylase activity which paralleled the loss of aromatase activity caused by these agents and which was antagonized by aromatase substrates. Conversely, 7-ethoxycoumarin antagonized the time-dependent action of 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione on aromatase and inhibited competitively the aromatization of 4-androstene-3,17-dione. The Ki for 7-ethoxycoumarin was equivalent to its Km as substrate for deethylation. It is concluded that a common oxidase species is responsible for both the aromatase and constitutive 7-ethoxycoumarin deethylase activities of human placental microsomes.     

Inhibition of human placental aromatase by mefloquine

Aromatase activity of human placental microsomes was inhibited competitively by the antimalarial drug, mefloquine, but not by the related drug, chloroquine. In the absence of any drug, the Km for testosterone was 47.1 +/- 2.3 nmol/l (mean +/- SD, n = 2). In the presence of chloroquine 500 mumol/l, the Km remained unchanged (47.4 +/- 1.8 nmol/l (mean +/- SD, n = 2), whereas mefloquine inhibited competitively with respect to substrate with a Ki value of 72 +/- 4.2 mumol/l (mean +/- SD, n = 2).     

A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity

Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.     

Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate

Cholesterol esterification by acyl CoA:cholesterol acyl transferase (ACAT) in macrophages is a key process in atheroma foam cell formation. However, the process of cholesterol substrate delivery to ACAT is not well defined. In this study, J774 macrophages, which form foam cells with native low density lipoprotein (LDL), were labeled with [3H]cholesterol-containing liposomes. Most (80-90%) of the cholesterol label could be converted by cholesterol oxidase to cholestenone, suggesting plasma membrane localization; only 0.6% of the label was in cholesteryl ester (CE). In cells chased for 6 h in medium lacking LDL, the distribution of label was essentially unchanged, whereas in cells chased with LDL, 28% of the label was incorporated into CE concomitant with a decrease in cholestenone label to 50%. [3H]Cholesterol-labeled mouse peritoneal macrophages incubated with acetyl-LDL, and both J774 and mouse peritoneal macrophages incubated with 25-hydroxy-cholesterol, also showed a shift of label from cholestenone to CE. Similar results were found when cellular cholesterol was biosynthetically labeled with [3H]mevalonate. The percentage of cholesterol substrate for ACAT in LDL-treated J774 macrophages which originates from endogenous cellular pools (versus that originating from LDL itself) is approximately 50%. We conclude that upon activation of ACAT in macrophages, there is a novel process whereby a cholesterol oxidase-accessible pool of cellular cholesterol, presumably plasma membrane cholesterol, is translocated to ACAT in the endoplasmic reticulum.     

Lipid involvement in oleoyl CoA desaturase activity of Fusarium oxysporum microsomes

Desaturation of oleoyl CoA by the microsomal fraction of Fusarium oxysporum hyphal cells required O2, NADPH, MgCl2, and the addition of either bovine serum albumin or the 105 000g supernatant fraction. In the absence of reduced nucleotide, [14C]oleoyl CoA was rapidly incorporated into phospholipid and triacylglycerol and hydrolyzed to free fatty acids. After addition of NADPH, oleate was desaturated at the normal rate. Analysis of the distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine and phosphatidylethanolamine were labeled with [14C]linoleate before any other lipid class. These results are consistent with oleoyl phospholipid being a direct intermediate in the desaturation of oleoyl CoA. The preference of the oleoyl-desaturase for NADPH, the relatively high pH optimum of 8.2, and the sensitivity to thenoyltrifluoroacetone inhibition suggest that some components of the microsomal electron-transport chain are common to both the oleoyl desaturase and stearoyl CoA desaturase systems in this fungus.