N-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, kills dopaminergic neurons after its accumulation in mitochondria where it inhibits Complex I of the respiratory chain. MPP+ inhibits respiration by binding to both a hydrophobic and a hydrophilic site on Complex I and this inhibition is increased by the lipophilic tetraphenylboron anion (TPB-) which facilitates movement of MPP+ through membranes and its penetration to the hydrophobic binding site on Complex I. To investigate the inhibition of respiration by MPP(+)-like compounds, we have measured simultaneously NADH-linked mitochondrial respiration and the uptake and accumulation of the N-benzyl-4-styrylpyridinium and N-ethyl-4-styrylpyridinium cations in mitochondria using ion-selective electrodes. The data provide direct evidence that TPB- increases the inhibition not by increasing matrix concentration but by facilitating access to the inhibitory sites on Complex I. We have also compared the rates of uptake of MPP+ analogues of varied lipophilicity by the inner membrane and the development of inhibition of NADH oxidation, using an inverted mitochondrial inner membrane preparation and appropriate ion-selective electrodes. These experiments demonstrated that the amount of MPP+ analogue bound to the inner membrane greatly exceeded the quantity required for complete inhibition of NADH oxidation. Moreover, binding to the membrane occurred much more rapidly than the development of inhibition with all MPP+ analogues tested. This suggests that the attainment of a correct orientation of these compounds within the membrane and the binding site may be a rate-limiting step in the development of inhibition.

The carnitine acyltransferases contribute to the modulation of the acyl-CoA/CoA ratio in various cell compartments with consequent effects on many aspects of fatty acid metabolism. The properties of the enzymes are different in each location. The kinetic mechanisms and kinetic parameters for the carnitine acyltransferases purified from peroxisomes (COT) and from the mitochondrial inner membrane (CPT-II) were determined. Product-inhibition studies established that COT follows a rapid-equilibrium random-order mechanism, but CPT-II follows a strictly ordered mechanism in which acyl-CoA or CoA must bind before the carnitine substrate. Hemipalmitoylcarnitinium [(+)-HPC], a prototype tetrahedral intermediate analogue of the acyltransferase reaction, inhibits CPT-II 100-fold better than COT. (+)-HPC behaves as an analogue of palmitoyl-L-carnitine with COT. In contrast, with CPT-II(+)-HPC binds more tightly to the enzyme than do substrates or products, suggesting that it is a good model for the transition state and, unlike palmitoyl-L-carnitine, (+)-HPC can bind to the free enzyme. The data support the concept of three binding domains for the acyltransferases, a CoA site, an acyl site and a carnitine site. The CoA site is similar in COT and CPT-II, but there are distinct differences between the carnitine-binding site which may dictate the kinetic mechanism.

Although the malonyl-CoA sensitivity of peroxisomal carnitine octanoyltransferase (COT) is reportedly lost on solubilization, we show that malonyl-CoA does inhibit the purified enzyme. Assay conditions such as buffer composition, pH, acyl-CoA substrate and the presence or absence of BSA can affect the observed inhibition. When assayed in the absence of BSA, COT shows simple competitive inhibition by malonyl-CoA. The Ki value for inhibition of purified COT is high (106 microM) compared with physiological concentrations (1-6 microM) and other short-chain acyl-CoA esters inhibit COT to the same degree. However, when COT is assayed in intact peroxisomes, the Ki for malonyl-CoA is almost 20-fold lower than found with the purified enzyme, whereas inhibition by other short-chain acyl-CoA esters does not change significantly. Several features of the inhibition of peroxisomal COT, including the specificity of malonyl-CoA over other short-chain acyl-CoA esters, resemble those of carnitine palmitoyltransferase (CPT)-I, suggesting that the regulation of COT and CPT-I in parallel may be necessary for the control of cellular fatty acid metabolism.

Salicylyl-CoA and benzoyl-CoA were good inhibitors of carnitine acetyltransferase (CAT), competing with acetyl-CoA with Ki values of 7.5 and 22 microM respectively in the forward direction and with CoA in the reverse reaction with similar Ki values. They were also competitive inhibitors of carnitine octanoyltransferase (Ki = 261 and 295 microM respectively), but were only weakly inhibitory to carnitine palmitoyltransferase. Inhibition of energy production by salicylate may result from the inhibition of CAT by salicylyl-CoA.

Carnitine palmitoyltransferase located in the erythrocyte plasma membrane is sensitive to inhibition by malonyl-CoA and 2-bromopalmitoyl-CoA plus carnitine. Although this inhibition and other properties suggest similarities to the intracellular enzymes in other tissues, no cross-reaction was observed with antisera to the peroxisomal or to the mitochondrial inner-membrane enzyme. The activity was solubilized by and was stable in Triton X-100, which destroys the enzymes found in microsomes and in the mitochondrial outer membrane. The substrate specificity is broader than for the intracellular enzymes, the activities with stearoyl-CoA (114%) and arachidonoyl-CoA (97%) being equal to that with palmitoyl-CoA, and the activities with linoleoyl-CoA (44%) and erucoyl-CoA (46%) about half that with palmitoyl-CoA. The function of this carnitine palmitoyltransferase is probably to buffer the acyl-CoA present in the erythrocyte for turnover of the fatty acyl groups of the membrane lipids.

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), interrupts mitochondrial electron transfer at the NADH dehydrogenase-ubiquinone junction, as do the respiratory chain inhibitors rotenone, piericidin A and barbiturates. Proof that these classical respiratory chain inhibitors and MPP+ react at the same site in the complex NADH dehydrogenase molecule has been difficult to obtain because none of these compounds bind covalently to the target. The 4′-alkyl derivatives of MPP+ inhibit NADH oxidation in submitochondrial particles at much lower concentrations than does MPP+ itself, but still dissociate on washing the membrane preparations, with consequent re-activation of the enzyme. The MPP+ analogues with short alkyl chains prevent the binding of 14C-labelled piericidin A to the membrane and thus must act at the same site, but analogues with alkyl chains longer than heptyl do not prevent binding of [14C]piericidin.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2′-ethyl- and 3′-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.

The activities of carnitine palmitoyltransferases (CPTs) of mitochondrial outer and inner membranes and of peroxisomes have been studied with carnitine analogues, namely DL-thiolcarnitine, DL-sulphocarnitine and L-aminocarnitine, using palmitoyl-CoA or octanoyl-CoA as co-substrate. With sulphocarnitine, both of the mitochondrial CPTs and the malonyl-CoA-sensitive CPT of peroxisomes showed appreciable activity with palmitoyl-CoA, but relatively lower activity when octanoyl-CoA was the co-substrate. The soluble CPT of peroxisomes did not show any activity with sulphocarnitine in the presence of either acyl-CoA. With thiolcarnitine, all of the CPTs showed more activity with palmitoyl-CoA than with octanoyl-CoA. None of the CPTs showed any activity with aminocarnitine and palmitoyl-CoA, but when the acyl donor was octanoyl-CoA, both of the malonyl-CoA-sensitive CPT enzymes showed considerable activity, unlike the malonyl-CoA-insensitive CPT isoenzymes. Aminocarnitine inhibited palmitoylcarnitine formation by both of the mitochondrial CPTs and by the CPT of gradient-purified peroxisomes, but the purified peroxisomal soluble CPT was not inhibited. These results show that the interaction of CPT enzymes with carnitine analogues, as substrates or inhibitors, is influenced by the chain length of the acyl-CoA substrate, and that the use of the appropriate carnitine analogue and acyl-CoA is likely to be useful for the discrimination of the various CPT activities in CPT deficiency disorders.

Inhibition of the overt mitochondrial carnitine palmitoyltransferase by malonyl-CoA is important in the regulation of fatty acid oxidation. In the past, the contribution of peroxisomal carnitine acyltransferase activity to the generation of medium- and long-chain acylcarnitines in the cytoplasm has been ignored. On the basis of marker enzyme levels, we now estimate that peroxisomal palmitoyltransferase activity constitutes about 20% of the peroxisomal plus overt-mitochondrial pool in fed rat liver. When assayed in situ, both the palmitoyltransferase and decanoyltransferase activities of gradient-purified peroxisomes are sensitive to malonyl-CoA, with up to 90% inhibition reached at less than 10 microM-malonyl-CoA. Very similar results were obtained with intact gradient-purified mitochondria from the same livers. In addition, the acyl-CoA substrate chain-length specificity was identical in both the peroxisomes and the mitochondria, with a decanoyltransferase/palmitoyltransferase ratio of 2. Thus the overt carnitine acyltransferase activities in peroxisomes and mitochondria have the same properties. Further, the malonyl-CoA sensitivity of the peroxisomal activity is lost on solubilization, as has been observed for the overt mitochondrial enzyme. It is suggested that malonyl-CoA inhibition of the peroxisomal enzyme as well as of the mitochondrial enzyme is important for the regulation of mitochondrial fatty acid oxidation.

The present studies examined the effect of agents that induce peroxisomal and mitochondrial beta-oxidation on hepatic mitochondrial carnitine palmitoyltransferase (CPT) and peroxisomal carnitine acyltransferase [CPTs of Ramsay (1988) Biochem. J. 249, 239-245; COT of Farrell & Bieber (1983) Arch. Biochem. Biophys. 222, 123-132 and Miyazawa, Ozasa, Osumi & Hashimoto (1983) J. Biochem. 94, 529-542]. In the first studies, high fat diets containing corn oil or fish oil were used to induce peroxisomal and mitochondrial enzymes. Rats were fed one of three diets for 4 weeks: (1) low fat, with corn oil as 11% of energy (kJ); (2) high fat, with corn oil as 45% of kJ; (3) high fat, with fish oil as 45% of kJ. At the end of 4 weeks, both mitochondrial CPT and peroxisomal CPTs exhibited increases in activity, immunoreactive protein, mRNA levels and transcription rates in livers of rats fed either high-fat diet compared to the low fat diet. Riboflavin deficiency or starvation for 48 h also increased the peroxisomal CPTs mRNA. A second set of studies used the plasticizer 2-(diethylhexyl)phthalate (DEHP), 0.5% clofibrate or 1% acetylsalicylic acid (fed for 3 weeks) to alter peroxisomal and mitochondrial fatty acid oxidation. With DEHP, the mitochondrial CPT and peroxisomal CPTs activity, immunoreactive protein, mRNA levels and and transcription rate were all increased by 3-5-fold. The peroxisomal CPTs activity, immunoreactive protein, mRNA levels and transcription rate were increased 2-3-fold by clofibrate and acetylsalicylic acid, again similar to mitochondrial CPT. The results of the combined studies using both diet and drugs to cause enzyme induction suggest that the synthesis of the carnitine acyltransferases (mitochondrial CPT and peroxisomal CPTs) may be co-ordinated with each other; however, the co-ordinate regulatory factors have not yet been identified.

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5′-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5′-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.

Electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF-Q oxidoreductase) catalyses the re-oxidation of reduced electron-transfer flavoprotein (ETF) with ubiquinone-1 (Q-1) as the electron acceptor. A kinetic assay for the enzyme was devised in which glutaryl-CoA in the presence of glutaryl-CoA dehydrogenase was used to reduce ETFox. and the reduction of Q-1 was monitored at 275 nm. The partial reactions involved in the overall assay system were examined. Glutaryl-CoA dehydrogenase catalyses the rapid reduction of ETFox. to the anionic semiquinone (ETF.-), but reduces ETF.- to the fully reduced form (ETFhq) at a rate that is about 6-fold lower. ETF.-, but not ETFhq, is directly re-oxidized by Q-1 at a rate that, depending on the steady-state concentration of ETF.-, may contribute significantly to the overall reaction. ETF-Q oxidoreductase catalyses rapid disproportionation of ETF.- with an equilibrium constant of about 1.0 at pH 7.8. In the presence of Q-1 it also catalyses the re-oxidation of ETFhq at a rate that is faster than that of the overall reaction. Rapid-scan experiments indicated the formation of ETF.-, but its fractional concentration in the early stages of the re-oxidation of ETFhq is low. The data indicate that the re-oxidation of ETFhq proceeds at a rate that is adequate to account for the overall rate of electron transfer from glutaryl-CoA to Q-1. An unusual property of ETF-Q oxidoreductase seems to be that it not only catalyses the re-oxidation of the reduced forms of ETF but also facilitates the complete reduction of ETFox. to ETFhq by disproportionation of the radical.

A novel technique for the separation of submitochondrial particles from the external medium, an essential procedure in transport studies, was devised. Very low concentrations of heparin (5-10 micrograms/ml) aggregate the particles and permit their rapid sedimentation in a micro-centrifuge. The transfer of activated fatty acids into mitochondria for oxidation depends on the exchange of matrix carnitine for external fatty-acylcarnitine. To study the matrix face of the carnitine/acylcarnitine translocase, inverted submitochondrial particles were prepared and loaded with L-[14C]carnitine. As found in intact mitochondria, the Km value for L-carnitine was 8 mM, that for palmitoyl-L-carnitine was two orders of magnitude lower, and 11-trimethylaminoundecanoyl-DL-carnitine was a competitive inhibitor. The properties of the carrier exposed to the outer and to the matrix sides of the mitochondrial inner membrane are thus similar.

Aconitase, as isolated from mammalian mitochondria by traditional methods, is virtually inactive and contains an oxidized [3Fe-4S]+ cluster. The activation of the enzyme and attendant conformational change have been studied by monitoring the changes in activity, in tryptophan fluorescence, and in the electron paramagnetic resonance of the cluster on incubation with dithionite, with and without added Fe2+. Restoration of the full activity is achieved with one electron per 3Fe cluster and at least 0.6 g-atoms of Fe2+ per mol. The process involves building up of [4Fe-4S]2+ clusters. Other metal ions do not substitute for Fe2+. Reduction alone, in the absence of added Fe2+, yields up to 70% of the maximum activity, but requires approx. 1.8 electrons of reductant per cluster. The results presented are consistent with the view that activation without added Fe2+ involves the destruction of some of the [3Fe-4S] clusters and the incorporation of the Fe so liberated into other clusters to yield a tetra-nuclear one. In particular, the effect of EDTA and of other iron chelators in inhibiting activation by dithionite alone is in accord with this view, although recent magnetic-circular-dichroism studies do not support this interpretation. The rates of increase in activity and tryptophan fluorescence are the same when Fe2+ is present, but in its absence, activation is very much slower than the increase in fluorescence, suggesting that the protein conformational change triggered by reduction of the Fe-S clusters precedes the insertion of the iron. Consistent with this view is the observation that iron chelators inhibit activation by dithionite, but not the increase in fluorescence and, hence, the conformational change. The results are discussed in light of data in the literature on the forms of the cluster and its possible function in catalysis.