Inhibition of acyl coenzyme A:lysolecithin acyltransferases by local anesthetics, detergents and inhibitors of cyclic nucleotide phosphodiesterases.

Acyl coenzyme A:lysolecithin acyltransferase plays a major role in regulating the amount of lysolecithin in cell membranes. The acyltransferase activity in microsomal preparations from rat liver, rat heart and rabbit gastric mucosa is inhibited by a series of tertiary amine local anesthetics, detergents, and some inhibitors of cyclic nucleotide phosphodiesterases. Aspirin and indomethacin cause elevated lysolecithin/lecithin ratios in the stomachs of mice after oral administration. Inhibition of acyltransferase activity in microsomal preparations by local anesthetics correlates with reported anesthetic potencies at approximately $\text{1}\text{100}$ reported therapeutic dosages. In BHK-13 cells acyltransferase activity is inhibited at $\text{1}\text{3}$ to $\text{1}\text{10}$ the concentrations that have been reported to cause alterations in the mobility and topography of cell surface receptors.     

Rimonabant is a dual inhibitor of acyl CoA:cholesterol acyltransferases 1 and 2

Acyl coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the intracellular synthesis of cholesteryl esters (CE). Both ACAT isoforms, ACAT1 and ACAT2, play key roles in the pathophysiology of atherosclerosis and ACAT inhibition retards atherosclerosis in animal models. Rimonabant, a type 1 cannabinoid receptor (CB1) antagonist, produces anti-atherosclerotic effects in humans and animals by mechanisms which are not completely understood. Rimonabant is structurally similar to two other cannabinoid receptor antagonists, AM251 and SR144528, recently identified as potent inhibitors of ACAT. Therefore, we examined the effects of Rimonabant on ACAT using both in vivo cell-based assays and in vitro cell-free assays. Rimonabant dose-dependently reduced ACAT activity in Raw 264.7 macrophages (IC₅₀ = 2.9 ± 0.38 μM) and isolated peritoneal macrophages. Rimonabant inhibited ACAT activity in intact CHO-ACAT1 and CHO-ACAT2 cells and in cell-free assays with approximately equal efficiency (IC₅₀ = 1.5 ± 1.2 μM and 2.2 ± 1.1 μM for CHO-ACAT1 and CHO-ACAT2, respectively). Consistent with ACAT inhibition, Rimonabant treatment blocked ACAT-dependent processes in macrophages, oxysterol-induced apoptosis and acetylated-LDL induced foam cell formation. From these results we conclude that Rimonabant is an ACAT1/2 dual inhibitor and suggest that some of the atherosclerotic beneficial effects of Rimonabant are, at least partly, due to inhibition of ACAT.     

Characterization of a bifunctional archaeal acyl coenzyme A carboxylase

Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an alpha(4)beta(4)gamma(4) subunit structure. The optimum temperature for the enzyme was 60 to 70 degrees C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent K(m) for acetyl-CoA was 0.17 +/- 0.03 mM, with a V(max) of 43.3 +/- 2.8 U mg(-1), and the K(m) for propionyl-CoA was 0.10 +/- 0.008 mM, with a V(max) of 40.8 +/- 1.0 U mg(-1). This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of Bacteria than to those of Eucarya. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit.     

Chemical events in chloropropionyl coenzyme A inactivation of acyl coenzyme A utilizing enzymes

Incubation of 3-chloropropionyl-CoA with 3-hydroxy-3-methylglutaryl-CoA synthase results in exchange of the C2 proton with solvent as inactivation of enzyme proceeds. This enzyme is also inhibited by S-acrylyl-N-acetylcysteamine; the limiting rate constant for inactivation by the acrylyl derivative (0.36 min-1) slightly exceeds the value measured for chloropropionyl-CoA (0.31 min-1). These observations support the intermediacy of acrylyl-CoA in the chloropropionyl-CoA-dependent inactivation of hydroxymethylglutaryl-CoA synthase. Inhibition of fatty acid synthase by chloropropionyl-CoA is primarily due to alkylation of a reactive cysteine, although secondary reaction with the enzyme's pantetheinyl sulfhydryl occurs. Modification of fatty acid synthase by S-acrylyl-N-acetylcysteamine occurs at a limiting rate (1.8 min-1) that is comparable to that estimated for chloropropionyl-CoA-dependent inactivation. However, this enzyme lacks the ability to deprotonate C2 of an acyl group such as the chloropropionyl moiety. Since such a step would be required to generate an acrylyl group from chloropropionyl-S-enzyme, it is likely that a typical affinity labeling process accounts for inactivation of fatty acid synthase by chloropropionyl-CoA. HMG-CoA lyase is also inhibited by S-acrylyl-N-acetylcysteamine. In contrast to the ability of this reagent to serve as a mechanism-based inhibitor of hydroxymethylglutaryl-CoA synthase and an affinity label of fatty acid synthase, it acts as a group-specific reagent in modifying HMG-CoA lyase (kappa 2 = 86.7 M-1 min-1).     

Fatty acyl coenzyme A-sensitive adenine nucleotide transport in a reconstituted liposome system

The adenine nucleotide translocase was purified from bovine heart mitochondria and incorporated into membranes of phospholipid liposomes. The rate of transport of the adenine nucleotides was competitively inhibited by oleoyl coenzyme A with an approximate Ki of 1.0 microM. Significant inhibition was limited to those fatty acyl coenzyme A esters which are carnitine dependent for their oxidation in isolated mitochondria. Octanoyl coenzyme A was almost completely inactive as was palmitic acid and palmitoyl carnitine. By comparing the inhibitory characteristics of carboxyatractylate and bongkrekic acid with those of oleoyl-CoA, it was determined that the fatty acyl-CoA esters could produce inhibition whether the carrier was inserted into the liposome in either the conventional (65%) or reverse (30%) orientation. The results demonstrate that the interaction of long chain fatty acyl-CoA esters with the ADP/ATP carrier in a purified reconstituted system mimics their effects with isolated mitochondria and inverted submitochondrial particles. In general, these findings are consistent with the role of acyl-CoA esters acting as natural ligands and biological effectors of the translocator.     

Inhibition of steryl glycoside biosynthesis by acyl coenzyme a and by digitonin

ATP, GTP, CoA, Mg²⁺, and Mn²⁺ did not inhibit biosynthesis of steryl glycoside and acylated steryl glycoside when added singly to enzyme preparations from spinach leaves. The combination of ATP (but not GTP), CoA, and Mg²⁺ or Mn²⁺ caused marked inhibition, especially of steryl glycoside biosynthesis, when reaction mixture concentrations of the additions were 0.2 millimolar. Inhibition was attributed to acyl-CoA and could be reproduced by palmitoyl-CoA. The inhibition could be partially prevented by bovine serum albumin. The effects of palmitoyl-CoA were distinct at 10 micromolar, and 50% inhibition of biosynthesis was observed at 40 micromolar.     

An accurate and versatile method for determining the acyl group-introducing position of lysophospholipid acyltransferases

Lysophospholipid acyltransferases (LPLATs) incorporate a fatty acid into the hydroxyl group of lysophospholipids (LPLs) and are critical for determining the fatty acid composition of phospholipids. Previous studies have focused mainly on their molecular identification and their substrate specificity regarding the polar head groups and acyl-CoAs. However, little is known about the positional specificity of the hydroxyl group of the glycerol backbone (sn-2 or sn-1) at which LPLATs introduce a fatty acid. This is mainly due to the instability of LPLs used as an acceptor, especially for LPLs with a fatty acid at the sn-2 position of the glycerol backbone (sn-2-LPLs), which are essential for the enzymatic assay to determine the positional specificity. In this study, we established a method to determine the positional specificity of LPLAT by preparing stable sn-2-LPLs in combination with PLA₂ digestion, and applied the method for determining the positional specificity of several LPLATs including LPCAT1, LYCAT and LPCAT3. We found that LPCAT1 introduced palmitic acid both at the sn-1 and sn-2 positions of palmitoyl-LPC, while LYCAT and LPCAT3 specifically introduced stearic acid at the sn-1 position of LPG and arachidonic acid at the sn-2 position of LPC, respectively. The present method for evaluating the positional specificity could also be used for biochemical characterization of other LPLATs.     

Spectroscopic analysis of the interaction of rat liver short-chain, medium-chain, and long-chain acyl coenzyme A dehydrogenases with acyl coenzyme A substrates

We systematically studied the visual spectral changes of short-chain, medium-chain, and long-chain acyl coenzyme A (acyl-CoA) dehydrogenases, purified from rat liver mitochondria, that occur upon reaction with acyl-CoA in the absence of an electron acceptor (half-reaction). Acyl-CoA esters having various chain lengths were tested, and changes in the steady-state spectral parameters were correlated with the turnover number in the complete reaction, which represented the ability of an enzyme/substrate combination to produce an enoyl-CoA. The long-wavelength absorbance, centered around 580 nm, was observed only in the enzyme/substrate combinations in which enoyl-CoA product was produced at a significant rate in the complete reaction. There was a good correlation between the magnitudes of the long-wavelength absorbance and the turnover numbers. In contrast, the bleaching of the flavin chromophore at 450 nm was observed not only in the titration with preferred substrates but also in that with unfavorable substrates, which were shorter than favorable substrates. In the interaction with the shorter than favorable substrates, however, enoyl-CoA was not produced, nor did long-wavelength absorbance occur. When short-chain and medium-chain acyl-CoA dehydrogenases were reacted with longer than favorable substrate from which no enoyl-CoA was produced, neither the appearance of the long-wavelength absorbance nor bleaching of flavin chromophore was observed. These data suggest that the catalytic base, which abstracts alpha-proton, and flavin adenine dinucleotide are internally located, and the region containing these two sites may physically be in the form of crevice or pocket.     

Structural requirements for charged lipid molecules to directly increase or suppress K+ channel activity in smooth muscle cells. Effects of fatty acids, lysophosphatidate, acyl coenzyme A and sphingosine

We determined the structural features necessary for fatty acids to exert their action on K+ channels of gastric smooth muscle cells. Examination of the effects of a variety of synthetic and naturally occurring lipid compounds on K+ channel activity in cell-attached and excised membrane patches revealed that negatively charged analogs of medium to long chain fatty acids (but not short chain analogs) as well as certain other negatively charged lipids activate the channels. In contrast, positively charged, medium to long chain analogs suppress activity, and neutral analogs are without effect. The key requirements for effective compounds seem to be a sufficiently hydrophobic domain and the presence of a charged group. Furthermore, those negatively charged compounds unable to "flip" across the bilayer are effective only when applied at the cytosolic surface of the membrane, suggesting that the site of fatty acid action is also located there. Finally, because some of the effective compounds, for example, the fatty acids themselves, lysophosphatidate, acyl Coenzyme A, and sphingosine, are naturally occurring substances and can be liberated by agonist- activated or metabolic enzymes, they may act as second messengers targeting ion channels.     

Micellization of fatty acyl-CoA mixtures and its relevance to the fatty acyl selectivity of acyltransferases

The critical micelle concentrations (CMCs) of palmitoyl-CoA/stearoyl-CoA and palmitoyl-CoA/oleoyl-CoA mixtures in 0.050 M KPi, pH 7.4, a buffer used in enzymatic studies, were determined by fluorescence. Mixed micelle solution theory, analogous to the thermodynamic treatment of vapor pressure, was applied to calculate monomer and micelle compositions. The behavior of the palmitoyl-CoA/stearoyl-CoA mixture is ideal, while the palmitoyl-CoA/oleoyl-CoA mixture, although not exhibiting ideal behavior, can be fitted reasonably well by nonideal theory. In both mixtures, selective micellization takes place and, unlike the case of pure fatty acyl-CoAs, above the CMC of the mixtures the concentration of molecules free in solution is strongly dependent upon total concentration. The information derived from the present physical studies becomes important in enzymatic studies with membrane-bound acyltransferases, where selectivity toward various fatty acyl donors, presented as binary mixtures, is frequently observed.     

Mutations in the midway gene disrupt a Drosophila acyl coenzyme A: diacylglycerol acyltransferase

During Drosophila oogenesis, defective or unwanted egg chambers are eliminated during mid-oogenesis by programmed cell death. In addition, final cytoplasm transport from nurse cells to the oocyte depends upon apoptosis of the nurse cells. To study the regulation of germline apoptosis, we analyzed the midway mutant, in which egg chambers undergo premature nurse cell death and degeneration. The midway gene encodes a protein similar to mammalian acyl coenzyme A: diacylglycerol acyltransferase (DGAT), which converts diacylglycerol (DAG) into triacylglycerol (TAG). midway mutant egg chambers contain severely reduced levels of neutral lipids in the germline. Expression of midway in insect cells results in high levels of DGAT activity in vitro. These results show that midway encodes a functional DGAT and that changes in acylglycerol lipid metabolism disrupt normal egg chamber development in Drosophila.     

Induction of acyl coenzyme A synthetase and hydroxyacyl coenzyme A dehydrogenase during fatty acid degradation in Neurospora crassa

Neurospora crassa is able to use long-chain fatty acids as the sole carbon and energy source. After growth on oleate there was nearly a 10-fold induction of the acyl coenzyme A (CoA) synthetase and a fivefold increase in the activity of the 3-hydroxyacyl-CoA dehydrogenase. There was a slight induction of the enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase, but no apparent induction of the flavin-linked acyl-CoA dehydrogenase. These noncoordinate changes in the fatty acid degradation enzymes suggest that they are not organized into a multienzyme complex as is found in bacteria.     

Properties of microsomal acyl coenzyme A reductase in mouse preputial glands

Synthesis of long-chain fatty alcohols in preputial glands of mice is catalyzed by an NADPH-dependent acyl coenzyme A (CoA) reductase located in microsomal membranes; sensitivity to trypsin digestion indicates that the reductase is on the cytoplasmic side of the membrane. Results with pyrazole and phenobarbital demonstrate the reaction is not catalyzed by a nonspecific alcohol dehydrogenase or an aldehyde reductase. Acyl-CoA reductase activity is sensitive to sulfhydryl and serine reagent modification, is stimulated by bovine serum albumin, and produces an aldehyde intermediate. The activity is extremely detergent sensitive and cannot be restored even after removal of the detergents. Phospholipase C or asolectin treatment does not release the acyl-CoA reductase from microsomal membranes, but causes a significant decrease in the activity recovered in the membrane pellet. Glycerol does not solubilize the reductase activity, nor does 3.0 m NaCl; however, the combination of glycerol and 3.0 m NaCl did release about 50% of the acyl-CoA reductase from the microsomal pellet. Substrate concentration curves obtained in the presence or absence of bovine serum albumin show significant differences in enzyme activities. The reductase is sensitive to the concentration of palmitoyl-CoA and is progressively inhibited at levels beyond the critical micellar concentration of the substrate. The apparent K_m for acyl-CoA reductase is 14 μm; however, the maximum velocity varies with the concentration of albumin used. Expression of enzyme activity in delipidated microsomes requires specific phospholipids, which suggests that in vivo regulation of acyl-CoA reductase activity could be achieved through modifications in membrane lipid composition.