Carnitine-acyltransferase activity of mitochondria from mung-bean hypocotyls

Carnitine-acyltransferase activity assayed with acetyl-CoA, octanoyl-CoA, or palmitoyl-CoA is associated with the mitochondrial but not with the peroxisomes of mung-bean hypocotyls. Using mitochondria as an enzyme source, a half-maximal reaction rate is obtained with a palmitoyl-CoA concentration approximately twice that required with acetyl-CoA. In the presence of a saturating acetyl-CoA concentration the carnitine-acyltransferase activity is not enhanced by palmitoyl-CoA as additional substrate. However, palmitoylcarnitine is formed in addition to acetylcarnitine, and the formation of acetylcarnitine is competitively inhibited by palmitoyl-CoA. It is concluded that the mitochondria of mung-bean hypocotyls possess a carnitine acyltransferase of broad substrate specificity with respect to the chainlength of the acyl-CoA and that the demonstration of a carnitine-palmitoyltransferase activity in plant mitochondria does not indicate the presence of a specific carnitine long-chain acyltransferase.     

Induction of peroxisomal enzymes in livers of neonatal rats exposed to lactating mothers treated with hypolipidaemic drugs. Role of drug metabolite transfer in milk.

Lactating rats were administered by gavage 100 mg/kg body wt. twice a day of either nafenopin or Wy-14,643, two hypolipidaemic drugs with hepatic peroxisome proliferative property. Neonatal rats, after feeding from the drug-treated mothers for 8-14 days, showed sustained increases in both the proliferation of hepatic peroxisomes, as well as in levels of the peroxisome-associated enzymes catalase (3-fold), carnitine acetyltransferase (15-35-fold), peroxisomal enoyl-CoA hydratase (29-46-fold), and palmitoyl-CoA oxidation (12-14-fold). These increases in enzyme activities in suckling rats were similar to those seen in the livers of the drug-treated, lactating mothers after 14 days of treatment. After administering [3H]nafenopin or [3H]Wy-14,643 to lactating rats, significant levels of drug-derived radioactivity were observed in suckling rat gastric milk curds by 2-4 h with significant radioactivity seen in suckling rat livers by 4-6 h. T.l.c. analysis of organic extracts of milk samples from [3H]Wy-14,643 treated animals indicated no detectable levels of the parent drug, only more-polar metabolites. Wy-14,643 metabolites preparatively purified from a rat liver microsomal fraction incubation induced peroxisome proliferation when injected into a neonatal rat. Preparative high pressure liquid chromatography purification and mass spectral analysis has allowed preliminary assessment of the structures of the Wy-14,643 microsomal metabolites. It is concluded that one or more of the metabolite fractions of Wy-14,643 transferred in milk exert the biological ability to induce peroxisome proliferation and peroxisomal enzymes in neonatal livers.     

The presence of peroxisomal carnitine palmitoyltransferase in chick embryo liver

Hepatic peroxisomes and mitochondria from 20-day-old chick embryo were separated by sucrose density gradient centrifugation and the characteristics of carnitine acyltransferases in these organelles were studied. The carnitine acyltransferase activities in peroxisomes were increased markedly by the treatment of chick embryo with clofibrate, while those in mitochondria did not change. In the liver of clofibrate-treated chick embryo, approximately 50% of total liver carnitine palmitoyltransferase (CPT) activity was present in the peroxisomal fraction. Peroxisomal CPT activity was easily solubilized, in contrast with mitochondrial CPT. The solubilized protein solutions from isolated peroxisomes and mitochondria were separately chromatographed on a column of Blue Sepharose CL-6B after the gel filtration on Sephadex G-25. Peroxisomal CPT was completely bound to a Blue Sepharose CL-6B column and was eluted below 0.25 M KCl, whereas mitochondrial CPT was not retained on the column. The substrate specificity profile of peroxisomal CPT with long-chain acyl-CoAs (C8 to C18) was similar to that of mitochondrial CPT, and the apparent Km value of peroxisomal CPT for palmitoyl-CoA was 5.2 microM, being similar to that of mitochondrial CPT. It is concluded that carnitine long-chain acyltransferase, which is different from mitochondrial CPT and is induced by clofibrate treatment, is present in peroxisomes of chick embryo liver.     

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria—case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria--case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Rat liver peroxisomes catalyze the beta oxidation of fatty acids

Peroxisomes were purified by differential and equilibrium density centrifugation from the livers of rats treated with clofibrate to enhance their peroxisomal system of fatty acid oxidation. These purified peroxisomes were tested for the presence of crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase using spectroscopic techniques that utilize the characteristic absorption bands of the appropriate 4-carbon acyl-CoA substrates. All three enzymes were found. Analysis of the fractions from equilibrium density centrifugation revealed major peaks of these enzyme activities in peroxisomes and excluded contamination by mitochondria as an explanation of the results. In the presence of excess CoA the purified peroxisomes oxidized palmitoyl-CoA to acetyl-CoA, and reduced NAD, with a 1:5:5 stoichiometry. The peroxisomes were inactive with butyryl-CoA and less active with octanoyl-CoA than with lauroyl-CoA or palmitoyl-CoA; they appear specialized for the beta oxidation of long chain fatty acids.     

Carnitine acyltransferase activities in rat brain mitochondria. Bimodal distribution, kinetic constants, regulation by malonyl-CoA and developmental pattern.

Carnitine palmitoyltransferase and carnitine octanoyltransferase activities in brain mitochondrial fractions were approx. 3-4-fold lower than activities in liver. Estimated Km values of CPT1 and CPT2 (the overt and latent forms respectively of carnitine palmitoyltransferase) for L-carnitine were 80 microM and 326 microM, respectively, and K0.5 values for palmitoyl-CoA were 18.5 microM and 12 microM respectively. CPT1 activity was strongly inhibited by malonyl-CoA, with I50 values (concn. giving 50% of maximum inhibition) of approx. 1.5 microM. In the absence of other ligands, [2-14C]malonyl-CoA bound to intact brain mitochondria in a manner consistent with the presence of two independent classes of binding sites. Estimated values for KD(1), KD(2), N1 and N2 were 18 nM, 27 microM, 1.3 pmol/mg of protein and 168 pmol/mg of protein respectively. Neither CPT1 activity, nor its sensitivity towards malonyl-CoA, was affected by 72 h starvation. Rates of oxidation of palmitoyl-CoA (in the presence of L-carnitine) or of palmitoylcarnitine by non-synaptic mitochondria were extremely low, indicating that neither CPT1 nor CPT2 was likely to be rate-limiting for beta-oxidation in brain. CPT1 activity relative to mitochondrial protein increased slightly from birth to weaning (20 days) and thereafter decreased by approx. 50%.     

Changes to the integral membrane protein composition of mouse liver peroxisomes in response to the peroxisome proliferators clofibrate,Wy-14,643 and Di(2-ethyl-hexyl)phthalate

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.     

Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism

Carnitine plays an essential role in mitochondrial fatty acid β-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.     

The medium-chain carnitine acyltransferase activity associated with rat liver microsomes is malonyl-CoA sensitive

The data presented herein show that both rough and smooth endoplasmic reticulum contain a medium-chain/long-chain carnitine acyltransferase, designated as COT, that is strongly inhibited by malonyl-CoA. The average percentage inhibition by 17 microM malonyl-CoA for 25 preparations is 87.4 +/- 11.7, with nine preparations showing 100% inhibition; the concentrations of decanoyl-CoA and L-carnitine were 17 microM and 1.7 mM, respectively. The concentration of malonyl-CoA required for 50% inhibition is 5.3 microM. The microsomal medium-chain/long-chain carnitine acyltransferase is also strongly inhibited by etomoxiryl-CoA, with 0.6 microM etomoxiryl-CoA producing 50% inhibition. Although palmitoyl-CoA is a substrate at low concentrations, the enzyme is strongly inhibited by high concentrations of palmitoyl-CoA; 50% inhibition is produced by 11 microM palmitoyl-CoA. The microsomal medium-chain/long-chain carnitine acyltransferase is stable to freezing at -70 degrees C, but it is labile in Triton X-100 and octylglucoside. The inhibition by palmitoyl-CoA and the approximate 200-fold higher I50 for etomoxiryl-CoA clearly distinguish this enzyme from the outer form of mitochondrial carnitine palmitoyltransferase. The microsomal medium-chain/long-chain carnitine acyltransferase is not inhibited by antibody prepared against mitochondrial carnitine palmitoyltransferase, and it is only slightly inhibited by antibody prepared against peroxisomal carnitine octanoyltransferase. When purified peroxisomal enzyme is mixed with equal amounts of microsomal activity and the mixture is incubated with the antibody prepared against the peroxisomal enzyme, the amount of carnitine octanoyltransferase precipitated is equal to all of the peroxisomal carnitine octanoyltransferase plus a small amount of the microsomal activity. This demonstrates that the microsomal enzyme is antigenically different than either of the other liver carnitine acyltransferases that show medium-chain/long-chain transferase activity. These results indicate that medium-chain and long-chain acyl-CoA conversion to acylcarnitines by microsomes in the cytosolic compartment is also modulated by malonyl-CoA.     

Active-site probes of carnitine acyltransferases. Inhibition of carnitine acetyltransferase by hemiacetylcarnitinium, a reaction intermediate analogue

Hemiacetylcarnitinium (2S,6R:2R,65)-6-carboxymethyl-2-hydroxy-2,4,4- trimethylmorpholinium) chloride is a relatively potent competitive inhibitor (Ki = 0.89 mM) of pigeon breast carnitine acetyltransferase (CAT) and of the crude rat liver CAT (Ki = 4.72 mM) but is neither an inhibitor nor an effective substrate for purified rat liver carnitine palmitoyltransferase (CPT). It does not inhibit state 3 oxygen consumption in isolated hepatic mitochondria using palmitoyl-CoA or palmitoylcarnitine as substrates. This compound is a reaction intermediate analogue of the proposed tetrahedral intermediate for acetyl transfer between acetylcarnitine and CoASH. Because the hemiketal carbon is chiral, a suggestion is made that one of the enantiomers has the same relative configuration as the proposed tetrahedral intermediate.     

Cloning, sequencing, and regulation of rat liver carnitine octanoyltransferase: transcriptional stimulation of the enzyme during peroxisome proliferation

Several complementary DNAs for the peroxisomal enzyme carnitine octanoyltransferase (COT), cloned in the expression vector lambda gt11, have been isolated. Together, these clones cover 2143 bp of the COT cDNA sequence with an open reading frame for 523 amino acids. Northern analysis showed the mRNA size for this enzyme to be 3.5 kilobases. The 523 residue long amino acid sequence amounts to a molecular mass of 60,269 daltons, indicating that the cloned cDNAs contain most or all of the coding sequence for COT (Mr approximately 62,000). Hybridization studies showed that the increased COT activity in the liver of rats, fed the potent peroxisome-proliferating drug Wy-14,643, is associated with a more than 40-fold rise in the steady-state level of the COT mRNA.     

Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p.

In Saccharomyces cerevisiae, beta-oxidation of fatty acids is confined to peroxisomes. The acetyl-CoA produced has to be transported from the peroxisomes via the cytoplasm to the mitochondrial matrix in order to be degraded to CO(2) and H(2)O. Two pathways for the transport of acetyl-CoA to the mitochondria have been proposed. The first involves peroxisomal conversion of acetyl-CoA into glyoxylate cycle intermediates followed by transport of these intermediates to the mitochondria. The second pathway involves peroxisomal conversion of acetyl-CoA into acetylcarnitine, which is subsequently transported to the mitochondria. Using a selective screen, we have isolated several mutants that are specifically affected in the second pathway, the carnitine-dependent acetyl-CoA transport from the peroxisomes to the mitochondria, and assigned these CDAT mutants to three different complementation groups. The corresponding genes were identified using functional complementation of the mutants with a genomic DNA library. In addition to the previously reported carnitine acetyl-CoA transferase (CAT2), we identified the genes for the yeast orthologue of the human mitochondrial carnitine acylcarnitine translocase (YOR100C or CAC) and for a transport protein (AGP2) required for carnitine transport across the plasma membrane.     

Effect of clofibrate on peroxisomal lignoceroyl-CoA ligase activity

The effect of a 2-week clofibrate (0.5%)-fortified diet on peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases was studied. The activities of palmitoyl-CoA and lignoceroyl-CoA ligases in peroxisomes isolated from clofibrate-treated animals were 4.4- and 4.0-fold higher than those of the controls. The different degrees of increases in these two enzyme activities support the previous conclusions that in peroxisomes palmitoyl-CoA ligase and lignoceroyl-CoA ligase are different enzymes. Since clofibrate treatment increases both of these peroxisomal acyl-CoA ligase activities and normal palmitoyl-CoA ligase is the source of the partial activity for the oxidation of lignoceric acid in X-ALD, treatment with a hypolipidemic drug, which can increase human peroxisomal enzyme activities, may be helpful in lowering the amount of the pathogen, VLC fatty acids, in X-ALD.     

Induction of fatty acid binding protein by peroxisome proliferators in primary hepatocyte cultures and its relationship to the induction of peroxisomal beta-oxidation

The induction of liver fatty acid binding protein (L-FABP) by the peroxisome proliferators bezafibrate and clofibrate was compared with the induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation in cultured rat hepatocytes maintained on a substratum of laminin-rich (EHS) gel. This substratum was chosen because marked induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was effected by bezafibrate in hepatocytes supported on EHS gel, whereas only peroxisomal palmitoyl-CoA oxidation was induced in hepatocytes maintained on collagen-coated plates. In control cells on EHS, activity of peroxisomal palmitoyl-CoA oxidation remained stable, while L-FABP abundance declined with time, and L-FABP mRNA was undetectable after 5 days. In cultures exposed to bezafibrate or clofibrate, peroxisomal palmitoyl-CoA oxidation activity was induced earlier and more rapidly than L-FABP. When fibrates were withdrawn, peroxisomal palmitoyl-CoA oxidation declined rapidly, whereas L-FABP continued to increase. L-FABP induction was accompanied by a striking increase in mRNA specifying this protein. Tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I, effectively doubled peroxisomal palmitoyl-CoA oxidation activity. However, tetradecylglycidic acid markedly inhibited fibrate induction of L-FABP and peroxisomal palmitoyl-CoA oxidation but, unexpectedly, did not prevent the fibrate-induced proliferation of peroxisomes. Maximal induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was produced at a bezafibrate concentration in the culture medium (0.05 mM) much lower than that of clofibrate (0.3 mM). Also, bezafibrate, but not clofibrate, inhibited [1-14C]oleic acid binding to L-FABP with a Ki = 9.5 microM. We conclude that hepatocytes maintained on EHS gel provide an important tool for investigating the regulation of L-FABP. These studies show that the induction of peroxisomal beta-oxidation and L-FABP by peroxisome proliferators are temporally consecutive but closely related processes which may be dependent on a mechanism distinct from that which leads to peroxisome proliferation. Furthermore, the mechanism of action of the more potent peroxisome proliferator, bezafibrate, may be mediated, in part, by interaction of this agent with L-FABP.     

Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver

The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.     

Peroxisome proliferation and induction of peroxisomal enzymes in mouse and rat liver by dehydroepiandrosterone feeding

Dehydroepiandrosterone (DHEA) treatment is effective in the prevention of various genetic and induced disorders of mice and rats. In studies designed to define some of the basic mechanisms that underline the beneficial chemopreventive effects exerted by the action of this steroid, we found that the liver undergoes profound changes that result in: (i) hepatomegaly; (ii) color change from pink to mahogany; (iii) proliferation of peroxisomes; (iv) increased cross-sectional area and volume density of peroxisomes; (v) increased or decreased number of mitochondria per cell; (vi) decreased mitochondrial cross-sectional area; (vii) marked induction of the peroxisomal bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; (viii) increased activities of enoyl-CoA hydratase and other peroxisomal enzymes assayed in this study, viz. catalase, carnitine acetyl-CoA transferase, carnitine octanoyl-CoA transferase, and urate oxidase; and (ix) increased activity of mitochondrial carnitine palmitoyl-CoA transferase. In addition, feeding DHEA to mice resulted in increased plasma cholesterol levels in two strains of mice evaluated in this study, and either slightly decreased or markedly increased plasma triglyceride levels, depending on the strain. Whether liver peroxisome proliferation, induced by DHEA feeding to mice and rats, plays a role in the chemopreventive effects elicited by this steroid remains to be established.     

Properties of purified carnitine acyltransferases of mouse liver peroxisomes

The purpose of this study was to characterize the physical, kinetic, and immunological properties of carnitine acyltransferases purified from mouse liver peroxisomes. Peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase were purified to apparent homogeneity from livers of mice fed a diet containing the hypolipidemic drug Wy-14,643 [( 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]-acetic acid). Both enzymes have a molecular weight of 60,000 and a similar pH optimum. Carnitine octanoyltransferase had a maximum activity for C6 moieties while the maximum for carnitine acetyltransferase was with C3 and C4 moieties. The apparent Km values were between 2 and 20 microM for the preferred acyl-CoA substrates, and the Km values for L-carnitine varied depending on the acyl-CoA cosubstrates used. The Hill coefficient, n, was approximately 1 for all acyl-CoAs tested, indicating Michaelis-Menten kinetics. Carnitine octanoyltransferase retained its maximum activity when preincubated with 5,5'-dithiobis-(2-nitrobenzoate) at pH 7.0 or 8.5. Neither carnitine octanoyltransferase nor carnitine acetyltransferase were inhibited by malonyl-CoA. The immunology of carnitine octanoyltransferase is discussed. These data indicate that peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase function in vivo in the direction of acylcarnitine formation, and suggest that the concentration of L-carnitine could influence the specificity for different acyl-CoA substrates.     

Metabolism of acetyl-CoA by isolated peroxisomal fractions: formation of acetate and acetoacetyl-CoA.

Liver peroxisomal fractions, isolated from rats treated with clofibrate, were shown to hydrolyze added [1-14C]acetyl-CoA to free [1-14C]acetate. [1-14C]Acetyl-CoA was, however, also converted to [14C]acetoacetyl-CoA. This reaction was inhibited by added ATP and by solubilization of the peroxisomes. The effect of ATP on synthesis of [14C]acetoacetyl-CoA was likely due to ATP-dependent stimulation of acetyl-CoA hydrolase (EC 3.1.2.1) activity. The inhibitory effect due to solubilizing conditions of incubation remains unexplained. During peroxisomal beta-oxidation of [1-14C]palmitoyl-CoA, [1-14C]acetyl-CoA, [1-14C]acetate, and [14C]acetoacetyl-CoA were shown to be produced. Possible metabolic implications of peroxisomal acetoacetyl-CoA synthesis are discussed.     

Effects of the mode of addition of acyl-CoA on the initial rate of formation of acylcarnitine in the presence of carnitine by intact rat liver mitochondria in vitro.

Time courses for the formation of palmitoylcarnitine from palmitoyl-CoA and carnitine, catalysed by the overt activity of carnitine palmitoyltransferase (CPT I) in rat liver mitochondria, were obtained. Significant initial non-linearity was observed only when reactions were started by addition of a concentrated solution of palmitoyl-CoA (4mM, to give a final concentration of 100 microM) uncomplexed to albumin. Minimal effects were observed when the reactions were started by addition of palmitoyl-CoA-albumin mixtures, even though the final palmitoyl-CoA/albumin molar ratios in the assay medium were identical in the two sets of experiments.