Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.     

Differential carnitine/acylcarnitine translocase expression defines distinct metabolic signatures in skeletal muscle cells

Import of acylcarnitine into mitochondrial matrix through carnitine/acylcarnitine-translocase (CACT) is fundamental for lipid catabolism. To probe the effect of CACT down-expression on lipid metabolism in muscle, human myocytes were stably transfected with CACT-antisense construct. In presence of low concentration of palmitate, transfected cells showed decreased palmitate oxidation and acetyl-carnitine content, increased palmitoyl-carnitine level, and reduced insulin-dependent decrease of fatty acylcarnitine-to-fatty acyl-CoA ratio. The augmented palmitoyl-carnitine synthesis, also in the presence of insulin, could be related to an altered regulation of carnitine-palmitoyl-transferase 1 (CPT 1) by malonyl-CoA, whose synthesis is dependent by the availability of cytosolic acetyl-groups. Indeed, all the described effects were completely overcome by CACT neo-expression by recombinant adenovirus vector or by addition of acetyl-carnitine to cultures. Acetyl-carnitine effect was related to an increase of malonyl-CoA and was abolished by down-expression, via antisense RNA strategy, of acetyl-CoA carboxylase-beta, the mitochondrial membrane enzyme involved in the direct CPT 1 inhibition via malonyl-CoA synthesis. Thus, in our experimental model the modulation of CACT expression has consequences for CPT 1 activity, while the biologic effects of acetyl-carnitine are not associated with a generic supply of energy compounds but to the anaplerotic property of the molecule.     

Regulation of palmitoyl-CoA inhibition of mitochondrial adenine nucleotide transport by cytosolic fatty acid binding protein.

Defatted liver fatty acid binding protein (FABP) reverses the inhibitory effect of palmitoyl-CoA on adenine nucleotide transport in rat liver mitochondria; addition of titrating amounts of FABP to mitochondria pretreated with palmitoyl-CoA stimulates nucleotide transport and that activation parallels the removal of the inhibitor from mitochondria. This effect is specific only for FABP; all other cytosolic proteins which do not bind fatty acids do not influence nucleotide transport activity. Addition of free fatty acids (which can compete for ligand binding sites on FABP) to mitochondria pretreated with palmitoyl-CoA interferes with the reversal activity of FABP. Adding FABP alone to freshly isolated mitochondria also activates nucleotide transport activity suggesting that the originally submaximal activity is probably due to the presence of endogenous long-chain acyl-CoA esters in the mitochondrial preparation. Because FABP is present in relatively high concentration in most mammalian cells, these observations offer a likely explanation of why the potent inhibitory effects of long-chain acyl-CoA esters on adenine nucleotide transport in isolated mitochondria are not seen in the intact cell.     

Fatty acids as acute regulators of the proton conductance of hamster brown-fat mitochondria

Possible mechanisms are evaluated for the acute regulation of the hamster brown-fat mitochondrial proton-conductance pathway which is active during non-shivering thermogenesis. Isolated mitochondria are incubated under conditions designed to approximate to the non-thermogenic state, and the effect of the steady infusion of fatty acids or acyl derivatives upon respiration, membrane potential and membrane proton conductance is monitored continuously. Fatty acids increase the proton conductance with no detectable threshold concentration, allowing the generated acyl carnitine to be rapidly oxidized. The extent of depolarization and of respiratory increase is a function of the rate of infusion. Immediately infusion is terminated the conductance decreases, the mitochondria repolarize and respiration returns to the initial rate. Infusion of acyl-CoA and acylcarnitine cause only a slight depolarization or respiratory increase after high concentrations of these derivatives have accumulated. Any factor which decreases the rate of conversion of fatty acid to acyl-CoA potentiates the conductance increase. An effect of acyl-CoA upon chloride permeability is not specific to brown-fat mitochondria. Fatty acids infused into rat liver mitochondrial incubations produced a small conductance increase, comparable to that of acyl-CoA or acylcarnitine. It is concluded that fatty acids are the most plausible acute regulators of the proton conductance. The relation to the brown-fat-specific 32000-Mr protein is discussed.     

Increase in mitochondrial content of long-chain acyl-CoA in brown adipose tissue during cold-acclimation

The mitochondrial content of long-chain acyl-CoA esters in the brown adipose tissue of guinea pigs increased 3.5-fold from a level of 92 +/- 17 pmol per mg protein (+/- S.E.; n = 7) in the control animals adapted at 22 degrees C to a new steady-state level of 328 +/- 20 pmol per mg protein (+/- S.E.; n = 46) after 10 days of cold-acclimation (5 degrees C). These low values of long-chain acyl-CoA species and the slow adaptive response for their increase do not support the proposal (Cannon, B., Sindin, U. and Romert, L. (1977) FEBS Lett. 4, 43-46) that the fatty acid CoA-esters have a physiological function in the regulation of the H+ (or OH-) permeability of the mitochondrial inner membrane. Experimental evidence is presented supporting the proposal that the long-chain acyl-CoA species are largely confined to the cytosolic side of the inner membrane. The activity of the adenine nucleotide translocase, as estimated at 25 degrees C in the reverse direction, was found to increase 5-fold upon depletion of the mitochondria of fatty acids (free and esterified) by preincubation with bovine serum albumin. The presence of potent inhibitors, i.e., long-chain acyl-CoA species, of adenine nucleotide translocation in brown adipose tissue of thermogenically active animals further supports the conclusion that ATP hydrolyzing mechanisms contribute insignificantly to long-term thermogenesis. The low values of long-chain acyl-CoA hydrolase (EC 3.1.2.1) activity, as measured in intact mitochondria and on a mitochondrial matrix fraction (i.e., 1.6 nmol X min-1 per mg protein), do not support the proposal that the hydrolase activity plays a significant role in the loose-coupling of brown adipose tissue mitochondria, either by a futile cycle mechanism or promoted by free fatty acid-induced uncoupling.     

Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion.

Several approaches were used to test the hypothesis proposing a role for acyl-CoA esters in nutrient-induced insulin release (Prentki, M., and Matschinsky, F. M. (1987) Physiol. Rev. 67, 1185-1248; Corkey, B. E., Glennon, M. C., Chen, K. S., Deeney, J. T., Matschinsky, F. M., and Prentki, M. (1989) J. Biol. Chem. 264, 21608-21612). Exogenous saturated long chain fatty acids markedly potentiated glucose-induced insulin release and elevated long chain acyl-CoA esters in the clonal beta-cell line (HIT). The secretory action depended on the fatty acid chain length, occurred in the range 3-20 microM (free concentration of palmitate), and was reversible and inhibitable by the neuromodulator somatostatin. 2-Bromopalmitate, an inhibitor of carnitine palmitoyl transferase I, suppressed the oxidation of endogenous fatty acids and promoted release of insulin. Only the nutrients or the combination of nutrients that caused secretion elevated malonyl-CoA. The short-chain acyl-CoA profile of HIT cells stimulated by various nutrients was determined in the presence of the nonstimulatory fuel glutamine. Glucose and leucine each provoked similar changes in acyl-CoA compounds. Both secretagogues elevated malonyl-CoA 3-6-fold, whereas succinyl-CoA, free CoASH, acetyl-CoA, and the free CoASH to acetyl-CoA ratio remained unaltered. Furthermore, only when inhibition of fatty acid oxidation was associated with a rise in malonyl-CoA did the total (mitochondrial plus cytoplasmic) content of long chain acyl-CoA esters correlate inversely with insulin release promoted by various nutrients. The results are consistent with the concept that fuel stimuli cause a rise in malonyl-CoA which by inhibiting fatty acid oxidation increase cytosolic long chain acyl-CoA esters. These data provide further support for a model in which malonyl-CoA and long chain acyl-CoAs esters serve as metabolic coupling factors when pancreatic beta-cells are stimulated with glucose and other nutrient secretagogues.     

Brain mitochondria are primed by moderate Ca2+ rise upon hypoxia/reoxygenation for functional breakdown and morphological disintegration

In animal models, brain ischemia causes changes in respiratory capacity, mitochondrial morphology, and cytochrome c release from mitochondria as well as a rise in cytosolic Ca2+ concentration. However, the causal relationship of the cellular processes leading to mitochondrial deterioration in brain has not yet been clarified. Here, by applying various techniques, we used isolated rat brain mitochondria to investigate how hypoxia/reoxygenation and nonphysiological Ca2+ concentrations in the low micromolar range affect active (state 3) respiration, membrane permeability, swelling, and morphology of mitochondria. Either transient hypoxia or a micromolar rise in extramitochondrial Ca2+ concentration, given as a single insult alone, slightly decreased active respiration. However, the combination of both insults caused devastating effects. These implied almost complete loss of active respiration, release of both NADH and cytochrome c, and rupture of mitochondria, as shown by electron microscopy. Mitochondrial respiration deteriorated even in the presence of cyclosporin A, documenting that membrane permeabilization occurred independent of mitochondrial permeability transition pore. Ca2+ has to enter the mitochondrial matrix in order to mediate this mitochondrial injury, because blockade of the mitochondrial Ca2+-transport system by ruthenium red in combination with CGP37157 completely prevented damage. Furthermore, protection of respiration from Ca2+-mediated damage by the adenine nucleotide ADP, but not by AMP, during hypoxia/reoxygenation is consistent with the delayed susceptibility of brain mitochondria to prolonged hypoxia, which is observed in vivo.     

Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.     

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.     

Control of fatty acid metabolism in ischemic and hypoxic hearts

The effects of whole heart ischemia on fatty acid metabolism were studied in the isolated, perfused rat heart. A reduction in coronary flow and oxygen consumption resulted in lower rates of palmitate uptake and oxidation to CO2. This decrease in metabolic rate was associated with increased tissue levels of long chain acyl coenzyme A and long chain acylcarnitine. Cellular levels of acetyl-CoA, acetylcarnitine, free CoA, and free carnitine decreased. These changes in CoA and its acyl derivatives indicate that beta oxidation became the limiting step in fatty acid metabolism. The rate of beta oxidation was probably limited by high levels of NADH and FADH2 secondary to a reduced supply of oxygen. Tissue levels of neutral lipids showed a slight increase durning ischemia, but incorporation of [U-14C]palmitate into lipid was not altered significantly. Although both substrates for lipid synthesis were present in higher concentrations during ischemia, compartmentalization of long chain acyl-CoA in the mitochondrial matrix and alpha-glycerol phosphate in the cytosol may have accounted for the relatively low rate of lipid synthesis.     

Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells.

Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.     

Inhibition of citrulline synthesis by octanoate and its modulation by adenine nucleotides

Liver mitochondria from octanoate-treated rabbits showed an impaired ability to synthesize citrulline. Two methods were used to evaluate citrulline synthesis in rat liver mitochondria. Under these conditions octanoate inhibited citrulline synthesis by over 50%. When ATP was included in the assay medium the inhibitory effect of octanoate was prevented. In the absence of ATP in the suspending medium, octanoate did not significantly lower total adenine nucleotides in rat liver mitochondria. However, under these conditions octanoate caused a change in the adenine nucleotide profile such that ATP content was decreased and AMP content was increased. When ATP was present in the assay medium, octanoate caused a similar increase in AMP content. However, ATP decreased only slightly. The alterations in mitochondrial adenine nucleotide profile by octanoate and the reversal of the effect by exogenous ATP suggests that octanoate inhibits citrulline synthesis via reduced intramitochondrial ATP levels. The ability of octanoate to lower mitochondrial ATP and elevate mitochondrial AMP may be related to its intramitochondrial activation by the medium chain fatty acid activating enzyme.     

Long-chain acylcarnitine content determines the pattern of energy metabolism in cardiac mitochondria

In the heart, a nutritional state (fed or fasted) is characterized by a unique energy metabolism pattern determined by the availability of substrates. Increased availability of acylcarnitines has been associated with decreased glucose utilization; however, the effects of long-chain acylcarnitines on glucose metabolism have not been previously studied. We tested how changes in long-chain acylcarnitine content regulate the metabolism of glucose and long-chain fatty acids in cardiac mitochondria in fed and fasted states. We examined the concentrations of metabolic intermediates in plasma and cardiac tissues under fed and fasted states. The effects of substrate availability and their competition for energy production at the mitochondrial level were studied in isolated rat cardiac mitochondria. The availability of long-chain acylcarnitines in plasma reflected their content in cardiac tissue in the fed and fasted states, and acylcarnitine content in the heart was fivefold higher in fasted state compared to the fed state. In substrate competition experiments, pyruvate and fatty acid metabolites effectively competed for the energy production pathway; however, only the physiological content of acylcarnitine significantly reduced pyruvate and lactate oxidation in mitochondria. The increased availability of long-chain acylcarnitine significantly reduced glucose utilization in isolated rat heart model and in vivo. Our results demonstrate that changes in long-chain acylcarnitine contents could orchestrate the interplay between the metabolism of pyruvate–lactate and long-chain fatty acids, and thus determine the pattern of energy metabolism in cardiac mitochondria.     

The effect of acute and prolonged ethanol treatment on the concentrations of coenzyme A, carnitine and their derivatives in rat liver

1. CoA, acetyl-CoA, long-chain acyl-CoA, carnitine, acetylcarnitine and long-chain acylcarnitine were measured in rat liver under various conditions. 2. Starvation caused an increase in the contents of these intermediates, except that of carnitine. 3. A single dose of ethanol had no effect on CoA content, whereas those of acetyl-CoA, acetylcarnitine and carnitine were increased and those of long-chain acyl-CoA and acylcarnitine were decreased. 4. Four weeks' adaptation to ethanol consumption did not change the effect of ethanol administration on these metabolites. 5. It is suggested that ethanol directly increases hepatic fatty acid synthesis and esterification. It is also suggested that this change is reversible and limited to the period of ethanol oxidation. 6. It is demonstrated that ethanol-induced triglyceride accumulation is not related to carnitine deficiency.     

Regulation of long chain fatty acid activation in heart muscle.

Regulation of fatty acid activation was studied in whole tissue homogenates of rat heart. The palmityl-CoA synthestase activity was proportional to the fatty acid to albumin ratio in the incubation medium with maximal activity occurring at a molar ratio of about 5. Fatty acyl-CoA synthetase activity was inhibited by products of the reaction (AMP, pyrophosphate, and palmityl-CoA). The apparent Ki for palmityl-CoA inhibition was 5 muM and this inhibition could be relieved by CoA-SH or albumin. The Km for CoA-SH in the absence of palmityl-CoA was 7 muM and was increased to 24 muM by addition of 8 muM palmityl-CoA. Cytosolic and mitochondrial levels of CoA-SH and carnitine were estimated in whole tissue homogenates of heart and liver. From 90 to 100% of whole tissue CoA was recovered in the mitochondrial fraction of heart muscle and it was estimated that the cytosolic concentration of free CoA-SH probably never exceeds its Km value for fatty acid activation in this tissue. Therefore, the rate of fatty acid activation would be expected to depend on the availability of CoA-SH in the cytosolic space. By adjusting the concentration of CoA-SH in the cytosol to the rate of acetyl-CoA oxidation, carnitineacetyl-CoA transferase may function in cardiac muscle to couple the rate of fatty acid activation in the cytosolic compartment to acetyl-CoA oxidation in the mitochondria. Approximately 30% of whole tissue CoA-SH was located in the cytosolic space in liver. Heart muscle has about twice as much carnitine as liver but in both tissues 100% of whole tissue carintine was located in the cytosolic space. The ratio of carnitine to CoA-SH in the cytosolic space was estimated to be about 100 in heart and 17 in liver. This high ratio in cardiac muscle may function to channel fatty acids toward oxidation rather than toward synthesis of complex lipids.     

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     

Energetic depression caused by mitochondrial dysfunction

Mitochondria not only provide most of the ATP needed for cell work and numerous specific anabolic and catabolic functions, they also contribute to Ca++ signalling and play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of the mt-genome or by acute processes, is responsible for numerous diseases. Decreased concentrations of adenine nucleotides, leaky outer and inner mitochondrial membranes, and decreased activities of respiratory chain enzymes contribute to depression of cellular energy metabolism, one of the most important consequences of mitochondrial impairment as characterized by decreased cytosolic phosphorylation potentials.     

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+).     

Fatty acid beta-oxidation in leukocytes from control subjects and medium-chain acyl-CoA dehydrogenase deficient patients.

In recent years an increasing number of inherited diseases in man have been identified in which there is an impairment in mitochondrial fatty acid oxidation. Diagnosis is usually done by gas-chromatographic analysis of urine, which may give difficulties, since urinary abnormalities may only be present intermittently. We therefore studied whether leukocytes could be used to study mitochondrial beta-oxidation directly. The results described herein show that leukocytes are able to beta-oxidize octanoate and palmitate. Furthermore, clear abnormalities in octanoate beta-oxidation were found in leukocytes from patients with an established deficiency of medium-chain acyl-CoA dehydrogenase, suggesting that measurement of octanoate and palmitate beta-oxidation in leukocytes may contribute to rapid diagnosis of medium-chain acyl-CoA dehydrogenase deficiency and presumably other mitochondrial beta-oxidation disorders.     

The influence of the cytosolic oncotic pressure on the permeability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the intermembrane space

Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.