Subcellular distribution of cholic acid:coenzyme a ligase and deoxycholic acid:Coenzyme a ligase activities in rat liver

Cholic acid:CoA ligase (EC 6.2.1.7, choloyl-CoA synthetase) and deoxycholic acid:CoA ligase catalyze the synthesis of choloyl-CoA and deoxycholoyl-CoA from their respective bile acids in rat liver. A modification of the phase partition assay was introduced which yields significantly (3-fold) higher specific activities for cholic acid:CoA ligase than previously reported. An independent method of separating choloyl-CoA from the substrates by high-pressure liquid chromatography was also developed and validates the modification. Both enzymic activities were found to be localized predominantly in the endoplasmic reticulum of rat liver. The level of either ligase in other purified, active subcellular fractions is consistent with the level of contamination by endoplasmic reticulum, estimated by using marker enzymes. Hence, the ligase assay can be used as a sensitive enzymic marker for endoplasmic reticulum in rat liver. The kinetic parameters of both enzymic activities were determined by using purified rough endoplasmic reticulum from rat liver. While the apparent maximal velocities for the two substrates are similar, the Michaelis constant for deoxycholate is significantly lower than that for cholate. Taurocholate and deoxycholate are shown to be competitive inhibitors of cholic acid:CoA ligase. The inhibition constant of deoxycholate is similar to its Michaelis constant for the deoxycholoyl-CoA-synthesizing reaction, suggesting that the same enzyme is responsible for both ligase activities.     

Characterization of an acyl-coA thioesterase that functions as a major regulator of peroxisomal lipid metabolism.

Peroxisomes function in beta-oxidation of very long and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. Here we have cloned and characterized a peroxisomal acyl-CoA thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA thioesterase 2). PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor alpha. Recombinant PTE-2 showed a broad chain length specificity with acyl-CoAs from short- and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA, and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acid-CoA:amino acid N-acyltransferase activity, suggesting that this thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.     

Participation of two members of the very long-chain acyl-CoA synthetase family in bile acid synthesis and recycling

Bile acids are synthesized de novo in the liver from cholesterol and conjugated to glycine or taurine via a complex series of reactions involving multiple organelles. Bile acids secreted into the small intestine are efficiently reabsorbed and reutilized. Activation by thioesterification to CoA is required at two points in bile acid metabolism. First, 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid, the 27-carbon precursor of cholic acid, must be activated to its CoA derivative before side chain cleavage via peroxisomal beta-oxidation. Second, reutilization of cholate and other C24 bile acids requires reactivation prior to re-conjugation. We reported previously that homolog 2 of very long-chain acyl-CoA synthetase (VLCS) can activate cholate (Steinberg, S. J., Mihalik, S. J., Kim, D. G., Cuebas, D. A., and Watkins, P. A. (2000) J. Biol. Chem. 275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. In contrast, VLCS activated 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate, but did not utilize any of the C24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C24 bile acids, whereas VLCS participates in the de novo synthesis pathway. Results of in situ hybridization, topographic orientation, and inhibition studies are consistent with the proposed roles of these enzymes in bile acid metabolism.     

Very long-chain acyl-CoA synthetases. Human "bubblegum" represents a new family of proteins capable of activating very long-chain fatty acids

Activation by thioesterification to coenzyme A is a prerequisite for most reactions involving fatty acids. Enzymes catalyzing activation, acyl-CoA synthetases, have been classified by their chain length specificities. The most recently identified family is the very long-chain acyl-CoA synthetases (VLCS). Although several members of this group are capable of activating very long-chain fatty acids (VLCFA), one is a bile acid-CoA synthetase, and others have been characterized as fatty acid transport proteins. It was reported that the Drosophila melanogaster mutant bubblegum (BGM) had elevated VLCFA and that the product of the defective gene had sequence homology to acyl-CoA synthetases. Therefore, we cloned full-length cDNA for a human homolog of BGM, and we investigated the properties of its protein product, hsBG, to determine whether it had VLCS activity. Northern blot analysis showed that hsBG is expressed primarily in brain. Compared with vector-transfected cells, COS-1 cells expressing hsBG had increased acyl-CoA synthetase activity with either long-chain fatty acid (2.4-fold) or VLCFA (2.6-fold) substrates. Despite this increased VLCFA activation, hsBG-expressing cells did not have increased rates of VLCFA degradation. Confocal microscopy showed that hsBG had a cytoplasmic localization in some COS-1 cells expressing the protein, whereas it appeared to associate with plasma membrane in others. Fractionation of these cells revealed that most of the hsBG-dependent acyl-CoA synthetase activity was soluble and not membrane-bound. Immunoaffinity-purified hsBG from transfected COS-1 cells was enzymatically active. hsBG and hsVLCS are only 15% identical, and comparison with sequences of two conserved motifs from all known families of acyl-CoA synthetases revealed that hsBG along with the D. melanogaster and murine homologs comprise a new family of acyl-CoA synthetases. Thus, two protein families are now known that contain enzymes capable of activating VLCFA. Because hsBG is expressed in brain but previously described VLCSs were not highly expressed in this organ, hsBG may play a central role in brain VLCFA metabolism and myelinogenesis.     

Human very long-chain acyl-CoA synthetase and two human homologs: initial characterization and relationship to fatty acid transport protein

Several human genes with a high degree of homology to rat very long-chain acyl-CoA synthetase (rVLCS) and mouse fatty acid transport protein (mFATP) were identified. Full-length cDNA clones were obtained for three genes, and predicted amino acid sequences were generated. Initial characterization indicated that one gene was most likely hVLCS, the human ortholog of rVLCS. The other two (hVLCS-H1 and hVLCS-H2) were more closely related to rVLCS than to mFATP. Phylogenetic analysis of amino acid sequences confirmed that hVLCS-H1 and hVLCS-H2 were evolutionarily closer to VLCSs than FATPs. Alignment of predicted amino acid sequences of human, rat and mouse VLCSs and FATPs revealed the existence of two highly conserved motifs. While one motif is also present in long-chain acyl-CoA synthetases, the other serves to distinguish the VLCS/FATP family from the long-chain synthetase family. Elucidation of the biochemical functions of all VLCS/FATP family members should provide new insights into cellular fatty acid metabolism.     

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [¹⁴C]ATP or [¹⁴C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg²⁺ and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [¹⁴C]ATP or with [³H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C₁₈ fatty acids.     

Acyl-Coenzyme A synthetase and fatty acid oxidation in rat liver peroxisomes.

Rat liver peroxisomes oxidized palmitate in the presence of ATP, CoA and NAD+, and the rate of palmitate oxidation exceeded that of palmitoyl-CoA oxidation. Acyl-CoA synthetase [acid: CoA ligase (AMP-forming); EC 6.2.1.3] was found in peroxisomes. The substrate specificity of the peroxisomal synthetase towards fatty acids with various carbon chain lengths was similar to that of the microsomal enzyme. The peroxisomal synthetase activity toward palmitate (40--100 nmol/min per mg protein) was higher than the rate of palmitate oxidation by the peroxisomal system (0.7--1.7 nmol/min per mg protein). The data show that peroxisomes activate long chain fatty acids and oxidize their acyl-CoA derivatives.     

The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using [1-14C]butyric acid and [1-14C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [14C]lignoceric acid into primary bile acids was approximately four times higher than that of [14C]butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo [F. Hashimoto and H. Hayashi (1987) Biochim. Biophys. Acta 921, 142-150]. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both [14C]lignoceric acid and [14C]butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.     

Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver

Short-, medium-, and long-chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short-chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 microM. The two mitochondrial xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases), HXM-A and HXM-B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K(I) values > 100 microM). These inhibitions were found to be the result of triacsin C competing with medium-chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long-chain fatty acid:CoA ligase (also termed long-chain fatty acyl-CoA synthetase, or long-chain acyl-CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high-affinity site with a K(I) of 0.1 microM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K(I) of 6 microM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high-affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site.The high-affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg(2+), with the EC(50) for inhibition being 3 mM free Mg(2+). The low affinity triacsin C inhibition was also enhanced by Mg(2+). The data suggests that Mg(2+) promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM-ligases by triacsin C, which showed only a low-affinity component, did not require Mg(2+).     

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Abstract: We measured long-chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl-CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.     

Human very-long-chain acyl-CoA synthetase: cloning, topography, and relevance to branched-chain fatty acid metabolism

Very-long-chain acyl-CoA synthetases (VLCS) activate very-long-chain fatty acids (VLCFA) containing 22 or more carbons to their CoA derivatives. We cloned the human ortholog (hVLCS) of the gene encoding the rat liver enzyme (rVLCS). Both hVLCS and rVLCS contain 620 amino acids, are expressed primarily in liver and kidney, and have a potential peroxisome targeting signal 1 (-LKL) at their carboxy termini. When expressed in COS-1 cells, hVLCS activated the VLCFA lignoceric acid (C24:0), a long-chain fatty acid (C16:0), and two branched-chain fatty acids, phytanic acid and pristanic acid. Immunofluorescence and immunoblot studies localized hVLCS to both peroxisomes and endoplasmic reticulum. In peroxisomes of HepG2 cells, hVLCS was topographically oriented facing the matrix and not the cytoplasm. This orientation, coupled with the observation that hVLCS activates branched-chain fatty acids, suggests that hVLCS could play a role in the intraperoxisomal reactivation of pristanic acid produced via alpha-oxidation of phytanic acid.     

Bile acid: CoASH ligases from guinea pig and porcine liver microsomes. Purification and characterization

A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed. Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids. Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay. The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction. The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e. ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr. A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer. The purified enzyme was extensively delipidated by adsorption to alumina. The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent. However, such additions did not enhance enzymatic activity. Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme. The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme. Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure. The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme. The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons. Thus, the functional form of the porcine enzyme also appears to be a dimer.     

Enzymatic and genetic characterization of firefly luciferase and Drosophila CG6178 as a fatty acyl-CoA synthetase

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.     

Sepharose-stearate as substrate for rat liver long-chain fatty acyl-CoA synthetase

Stearic acid coupled covalently to Sepharose 6B serves as substrate for thioesterification catalyzed by rat liver long-chain fatty acyl-CoA synthetase (ATP-forming) (EC 6.2.1.3). Availability as substrate is dependent upon the conservation of the free omega-terminal in addition to that of the free carboxyl function. The enzymatic overall formation of matrix-acyl-CoA in the presence of ATP and CoA as cosubstrates conforms to the stoichiometry reported for thioesterification of the free long-chain fatty acyl substrate. The preformed matrix-acyl-CoA serves as substrate for the backward synthetase reaction in the presence of AMP and PPi. The apparent Km values for ATP and CoA in the presence of the acyl matrix are similar to the respective Km values observed in the presence of the free acid substrate. The apparent Km for the acyl matrix is 10-fold higher (0.5 mM) than the apparent Km value for the free acid. The feasibility of enzymatic thioesterification of bound long-chain fatty acids implies that the exact nature of the bulky chain situated between the carboxy and omega-terminal plays a secondary role in defining the fatty acyl substrate specificity for long-chain fatty acyl-CoA synthetase. Also, dissociation of bound long-chain fatty acids does not constitute an obligatory preliminary step to fatty acid thioesterification.     

Measurement and subcellular distribution of choloyl-CoA synthetase and bile acid-CoA:amino acid N-acyltransferase activities in rat liver

An improved method for assaying choloyl-CoA synthetase activity (E.C. 6.2.1.7) and two methods for specific measurement of bile acid-CoA:amino acid N-acyltransferase activity (E.C. 2.3.1) are described. The methods are shown to be reproducible, linear with respect to time and enzyme protein, and result in estimates of enzymic activity that conform to the theoretical stoichiometry of the individual reactions. Utilizing these methods, the subcellular distribution of the rat liver enzymic activity catalyzing the formation of glycine and taurine conjugates of bile acids is shown. Choloyl-CoA synthetase is associated with the microsomal membranes and bile acid-CoA:amino acid N-acyltransferase activity with the postmicrosomal supernatant. No significant amino acid N-acyltransferase activity is present in the lysosome fraction. These studies provide methods that will permit further study of the individual enzymic reactions involved in the intrahepatic conjugation of bile acids with amino acids.     

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.     

Characteristics and subcellular localization of pristanoyl-CoA synthetase in rat liver.

We have investigated the activation of pristanic acid to its CoA-ester in rat liver. The results show that peroxisomes, mitochondria as well as microsomes contain pristanoyl-CoA synthetase activity. On the basis of competition experiments and immunoprecipitation studies using antibodies raised against rat liver microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3) we conclude that pristanic acid is activated by the same enzyme which activates long-chain fatty acids, i.e., long-chain fatty acyl-CoA synthetase.     

Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver.

The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.