High-affinity binding of fatty acyl-CoAs and peroxisome proliferator-CoA esters to glutathione S-transferases effect on enzymatic activity.

Acyl-CoAs are present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl-CoA concentration, compatible with their metabolic role, their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl-CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl-CoA binding protein (ACBP) and fatty acyl-CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl-CoAs. Whether these proteins also bind xenobiotic-CoA is not known. Here we have identified new liver cytosolic fatty acyl-CoA and xenobiotic-CoA binding sites as glutathione S-transferase (GST), using fluorescent polarization and a acyl-etheno-CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1-1, GSTP1-1 and GSTM1-1 were used. Only class alpha rat liver GST and human GSTA1-1 bind xenobiotic-CoAs and fatty acyl-CoAs, with Kd values ranging from 200 nM to 5 microM. One mol of acyl-CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat liver GST and human recombinant GSTA1-1 (IC50 at the nanomolar level for palmitoyl-CoA) but not GSTP1-1 and GSTM1-1. Acyl-CoAs do not interact with the GSTA1-1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl-CoA concentration, as occurs after exposure to peroxisome proliferators, acyl-CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification. Analysis of the binding properties of GSTs and other acyl-CoA binding proteins suggest that under increased acyl-CoA concentration GSTs would be responsible for xenobiotic-CoA binding whereas ACBP would preferentially bind fatty acyl-CoAs.     

A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1

Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatis mc(2)155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinant Pseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.     

Acyl-CoA-binding protein (ACBP) and its relation to fatty acid-binding protein (FABP): an overview

Summary Acyl-CoA-binding protein is a 10 Kd protein which binds medium- and long-chain acyl-CoA esters with high affinity. The concentration in liver is 2–4 times the acyl-CoA concentration. ACBP has much greater affinity for acyl-CoA than FABP. FABP from bovine heart and liver is unable to compete with multilamellar liposomes, Lipidex and microsomal membrane in binding acyl-CoA esters, whereas ACBP effectively extracts acyl-CoA from all those sources. Previously published results on the effect of FABP on acyl-CoA metabolism need to be reevaluated due to possible contamination with ACBP. Recently it was discovered that ACBP is identical to a putative neurotransmitter diazepam binding inhibitor. The possibility therefore exists that ACBP has more than one function.     

Long-chain acyl-CoA esters and acyl-CoA binding protein are present in the nucleus of rat liver cells

A detailed analysis of the subcellular distribution of acyl-CoA esters in rat liver revealed that significant amounts of long-chain acyl-CoA esters are present in highly purified nuclei. No contamination of microsomal or mitochondrial marker enzymes was detectable in the nuclear fraction. C16:1 and C18:3-CoA esters were the most abundant species, and thus, the composition of acyl-CoA esters in the nuclear fraction deviates notably from the overall composition of acyl-CoA esters in the cell. After intravenous administration of the non-beta-oxidizable [(14)C]tetradecylthioacetic acid (TTA), the TTA-CoA ester could be recovered from the nuclear fraction. Acyl-CoA esters bind with high affinity to the ubiquitously expressed acyl-CoA binding protein (ACBP), and several lines of evidence suggest that ACBP functions as a pool former and transporter of acyl-CoA esters in the cytoplasm. By using immunohistochemistry, immunofluorescence microscopy, and immunoelectron microscopy we demonstrate that ACBP localizes to the nucleus as well as the cytoplasm of rat liver cell and rat hepatoma cells, suggesting that ACBP may also be involved in regulation of acyl-CoA-dependent processes in the nucleus.     

Assays of protein palmitoylation

Protein palmitoylation plays an important role in the structure and function of a wide array of proteins. Unlike other lipid modifications, protein palmitoylation is highly dynamic and cycles of palmitoylation and depalmitoylation can regulate protein function and localization. The dynamic nature of palmitoylation is poorly resolved because of limitations in assay methods. Here, we discuss various methods that can be used to measure protein palmitoylation and identify sites of palmitoylation. We describe new methodology based on "fatty acyl exchange labeling" in which palmitate is removed via hydroxylamine-mediated cleavage of the palmitoyl-thioester bond and then exchanged with a sulfhydryl-specific labeling compound. The techniques are highly sensitive and allow for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or non-radioactive, and can be used to assay the palmitoylation of proteins from tissue samples.     

Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development

Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein–lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16- to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (β-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds.     

Comparison of the binding affinities of acyl-CoA-binding protein and fatty-acid-binding protein for long-chain acyl-CoA esters.

Bovine and rat liver acyl-CoA-binding proteins (ACBP) were found to exhibit a much higher affinity for long-chain acyl-CoA esters than both bovine hepatic and cardiac fatty-acid-binding proteins (hFABP and cFABP respectively). In the Lipidex 1000- as well as the liposome-binding assay, bovine and rat hepatic ACBP effectively bound long-chain acyl-CoA ester, h- and c-FABP were, under identical conditions, unable to bind significant amounts of long-chain acyl-CoA esters. When FABP, ACBP and [1-14C]hexadecanoyl-CoA were mixed, hexadecanoyl-CoA could be shown to be bound to ACBP only. The experimental results give strong evidence that ACBP, and not FABP, is the predominant carrier of acyl-CoA in liver.     

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).     

A novel acyl-CoA-binding protein from bovine liver. Effect on fatty acid synthesis.

Bovine liver was shown to contain a hitherto undescribed medium-chain acyl-CoA-binding protein. The protein co-purifies with fatty-acid-binding proteins, but was, unlike these proteins, unable to bind fatty acids. The protein induced synthesis of medium-chain acyl-CoA esters on incubation with goat mammary-gland fatty acid synthetase. The possible function of the protein is discussed.     

Global analysis of protein palmitoylation in African trypanosomes

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.     

DHHC20 Palmitoyl-Transferase Reshapes the Membrane to Foster Catalysis

Cysteine palmitoylation, a form of S-acylation, is a key posttranslational modification in cellular signaling. This type of reversible lipidation occurs in both plasma and organellar membranes, and is catalyzed by a family of integral membrane proteins known as DHHC acyltransferases. The first step in the S-acylation process is the recognition of free acyl coenzyme A (acyl-CoA) from the lipid bilayer. The DHHC enzyme then becomes autoacylated at a site defined by a conserved Asp-His-His-Cys motif. This reaction entails ionization of the catalytic Cys. Intriguingly, in known DHHC structures, this catalytic Cys appears to be exposed to the hydrophobic interior of the lipid membrane, which would be highly unfavorable for a negatively charged nucleophile, thus hindering autoacylation. Here, we use biochemical and computational methods to reconcile these seemingly contradictory facts. First, we experimentally demonstrate that human DHHC20 is active when reconstituted in POPC nanodiscs. Microsecond-long all-atom molecular dynamics simulations are then calculated for human DHHC20 and for different acyl-CoA forms, also in a POPC membrane. Strikingly, we observe that human DHHC20 induces a drastic deformation in the membrane, particularly on the cytoplasmic side, where autoacylation occurs. As a result, the catalytic Cys becomes hydrated and optimally positioned to encounter the cleavage site in acyl-CoA. In summary, we hypothesize that DHHC enzymes locally reshape the membrane to foster a morphology that is specifically adapted for acyl-CoA recognition and autoacylation.     

The macrophage-induced gene (mig) of Mycobacterium avium encodes a medium-chain acyl-coenzyme A synthetase.

The macrophage-induced gene (mig) of Mycobacterium avium has been associated with virulence, but the functions of the gene product were still unknown. Here we have characterized the Mig protein by biochemical methods. A plasmid with a histidine-tagged fusion protein was constructed for expression in Escherichia coli. Mig was detected as a 60 kDa protein after expression and purification of the recombinant gene product. The sequence of the fusion gene and of the parent gene in M. avium were reexamined. This confirmed that the mig gene encodes a 550 amino acid protein (58 kDa) instead of a 295 amino acid protein (30 kDa) as predicted before. The 550 amino acid Mig exhibits a high degree of homology to bacterial acyl-CoA synthetases. Two artificial 30 kDa derivatives of Mig were expressed and purified as histidine-tagged fusion proteins in E. coli. These proteins and the 58.6 kDa histidine-tagged Mig protein were analysed for activity with an acyl-CoA synthetase assay. Among the three investigated proteins, only the 58.6 kDa Mig exhibited detectable activity as an acyl-CoA synthetase (EC 6.2.1.3) with saturated medium-chain fatty acids, unsaturated long-chain fatty acid and some aromatic carbon acids as substrates. Enzymatic activity could be inhibited by 2-hydroxydodecanoic acid, a typical inhibitor of medium-chain acyl-CoA synthetases. We postulate a novel medium-chain acyl-CoA synthetase motif. We have investigated the biochemical properties of Mig and suggest that this enzyme is involved in the metabolism of fatty acid during mycobacterial survival in macrophages.     

Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation

Protein palmitoylation is a post-translational modification that affects a great number of proteins. In most cases, the enzymes responsible for this modification have not been identified. Some proteins use palmitoylation to attach themselves to membranes; however, palmitoylation also occurs in transmembrane proteins, and the function of this palmitoylation is less clear. Here we identify Swf1, a member of the DHHC-CDR family of palmitoyltransferases, as the protein responsible for modifying the yeast SNAREs Snc1, Syn8 and Tlg1, at cysteine residues close to the cytoplasmic end of their single transmembrane domains (TMDs). In an swf1Delta mutant, Tlg1 is mis-sorted to the vacuole. This occurs because unpalmitoylated Tlg1 is recognised by the ubiquitin ligase Tul1, resulting in its targeting to the multivesicular body pathway. Our results suggest that one role of palmitoylation is to protect TMDs from the cellular quality control machinery, and that Swf1 may be the enzyme responsible for most, if not all, TMD-associated palmitoylation in yeast.     

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).     

Palmitoyl acyltransferase assays and inhibitors (Review)

Palmitoylated proteins have been implicated in several disease states including Huntington's, cardiovascular, T-cell mediated immune diseases, and cancer. To proceed with drug discovery efforts in this area, it is necessary to: identify the target enzymes, establish efficient assays for palmitoylation, and conduct high-throughput screening to identify inhibitors. The primary objectives of this review are to examine the types of assays used to study protein palmitoylation and to discuss the known inhibitors of palmitoylation. Six main palmitoylation assays are currently in use. Four assays, radiolabeled palmitate incorporation, fatty acyl exchange chemistry, MALDI-TOF MS and azido-fatty acid labeling are useful in the identification of palmitoylated proteins and palmitoyl acyltransferase (PAT) enzymes. Two other methods, the in vitro palmitoylation (IVP) assay and a cell-based peptide palmitoylation assay, are useful in the identification of PAT enzymes and are more amenable to screening for inhibitors of palmitoylation. To date, two general types of palmitoylation inhibitors have been identified. Lipid-based palmitoylation inhibitors broadly inhibit the palmitoylation of proteins; however, the mechanism of action of these compounds is unknown, and each also has effects on fatty acid biosynthesis. Conversely, several non-lipid palmitoylation inhibitors have been shown to selectively inhibit the palmitoylation of different PAT recognition motifs. The selective nature of these compounds suggests that they may act as protein substrate competitors, and may produce fewer non-specific effects. Therefore, these molecules may serve as lead compounds for the further development of selective inhibitors of palmitoylation, which may lead to new therapeutics for cancer and other diseases.     

Effect of ATP Depletion on the Palmitoylation of Myelin Proteolipid Protein in Young and Adult Rats

The present study was designed to determine whether the palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is dependent on cellular energy. To this end, brain slices from 20- and 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various metabolic poisons. In adult rats, the inhibition of mitochondrial ATP production with KCN (5 mM), oligomycin (10 microM), or rotenone (10 microM) reduced the incorporation of [3H]palmitate into fatty acyl-CoA and glycerolipids by 50-60%, whereas the labeling of PLP was unaltered. Incubation in the presence of rotenone (10 microM) plus NaF (5 mM) abolished the synthesis of acyl-CoA and lipid palmitoylation, but the incorporation of [3H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP-dependent and ATP-independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP-dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP-independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.     

Microbial formation of esters

Small aliphatic esters are important natural flavor and fragrance compounds and have numerous uses as solvents and as chemical intermediates. Besides the chemical or lipase-catalyzed formation of esters from alcohols and organic acids, small volatile esters are made by several biochemical routes in microbes. This short review will cover the biosynthesis of esters from acyl-CoA and alcohol condensation, from oxidation of hemiacetals formed from aldehydes and alcohols, and from the insertion of oxygen adjacent to the carbonyl group in a straight chain or cyclic ketone by Baeyer–Villiger monooxygenases. The physiological role of the ester-forming reactions can allow degradation of ketones for use as a carbon source and may play a role in detoxification of aldehydes or recycling cofactors. The enzymes catalyzing each of these processes have been isolated and characterized, and a number of genes encoding the proteins from various microbes have been cloned and functionally expressed. The use of these ester-forming organisms or recombinant organisms expressing the appropriate genes as biocatalysts in biotechnology to make specific esters and chiral lactones has been studied in recent years.     

Chemical and genetic probes for analysis of protein palmitoylation

Reversible protein palmitoylation is one of the most important posttranslational modifications that has been implicated in the regulation of protein signaling, trafficking, localizing and enzymatic activities in cells and tissues. In order to achieve a precise understanding of mechanisms and functions of protein palmitoylation as well as its roles in physiological processes and disease progression, it is necessary to develop techniques that can qualitatively and quantitatively monitor the dynamic protein palmitoylation in vivo and in vitro. This review will highlight recent advances in both chemical and genetic encoded probes that have been developed for accurate analysis of protein palmitoylation, including identification and quantification of acyl moieties and palmitoylated proteins, localization of amino acid residues on which acyl moieties are attached, and imaging of cellular distributions of palmitoylated proteins. The role of major techniques of fluorescence microscopy and mass spectrometry in facilitating the analysis of protein palmitoylation will also be explored.     

Global analysis of protein palmitoylation in yeast

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal