The presence of acyl-CoA hydrolase in rat brown-adipose-tissue peroxisomes.

The subcellular distribution of acyl-CoA hydrolase was studied in rat brown adipose tissue, with special emphasis on possible peroxisomal localization. Subcellular fractionation by sucrose-density-gradient centrifugation, followed by measurement of short-chain (propionyl-CoA) acyl-CoA hydrolase in the presence of NADH, resulted in two peaks of activity in the gradient: one peak corresponded to the distribution of cytochrome oxidase (mitochondrial marker enzyme), and another peak of activity coincided with the peroxisomal marker enzyme catalase. The distribution of the NADH-inhibited short-chain hydrolase activity fully resembled that of cytochrome oxidase. The substrate-specificity curve of the peroxisomal acyl-CoA hydrolase activity indicated the presence of a single enzyme exhibiting a broad substrate specificity, with maximal activity towards fatty acids with chain lengths of 3-12 carbon atoms. The mitochondrial acyl-CoA hydrolase substrate specificity, in contrast, indicated the presence of at least two acyl-CoA hydrolases (of short- and medium-chain-length specificity). The peroxisomal acyl-CoA hydrolase activity was inhibited by CoA at low (microM) concentrations and by ATP at high concentrations (greater than 0.8 mM). In contrast with the mitochondrial short-chain hydrolase, the peroxisomal acyl-CoA hydrolase activity was not inhibited by NADH.     

NADH-sensitive propionyl-CoA hydrolase in brown-adipose-tissue mitochondria of the rat

Acyl-CoA hydrolase activity was studied in brown adipose tissue (BAT) mitochondria of rats. The substrate specificity was investigated: total hydrolase activity showed two activity peaks, one sharp peak for propionyl-CoA and a broad peak at medium- to long-chain acyl-CoAs. The propionyl-CoA activity fully comigrated with a mitochondrial matrix marker enzyme in fractionation studies of tissue and mitochondria. The hydrolytic activity against short-chain acyl-CoAs was inhibited by NADH, and analyses of the substrate specificity of the hydrolases in the presence and absence of NADH allowed for the delineation of two distinct acyl-CoA hydrolases. These hydrolases could also be separated by gel filtration. It was concluded that rat BAT mitochondria possess at least two matrix acyl-CoA hydrolases: one broad-spectrum acyl-CoA hydrolase with an apparent native molecular weight of less than 100,000, and a specific propionyl-CoA hydrolase with an apparent native molecular weight at least 240,000; this hydrolase is regulated by NADH. It is suggested that the function of the propionyl-CoA hydrolase is to ensure that the level of propionyl-CoA in the mitochondria is not detrimentally increased.     

Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-L-carnitine hydrolase from rat liver.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.     

Intracellular localization of long-chain acyl-coenzyme A hydrolase and acyl-L-carnitine hydrolase in brown adipose tissue from guinea pigs.

The activities of long-chain acyl-CoA hydrolase (palmitoyl-CoA hydrolase, EC 3.1.2.2) and long-chain acyl-L-carnitine hydrolase, EC 3.1.1.28) in brown adipose tissue from cold-exposed and control guinea pigs were studied. Mitochondria from cold-exposed animals hydrolysed 21 nmol of palmitoyl-CoA/min per mg of protein and 1.3 nmol of palmitoyl-L-carnitine/min per mg of protein, and the specific activities were respectively 2 and 5 times as high in cold-exposed as in control animals. The subcellular-localization studies showed that both the long-chain acyl-CoA hydrolase and long-chain acyl-L-carnitine hydrolase were localized in the mitochondria. A location also in the soluble fraction cannot be excluded. The long-chain acyl-CoA hydrolase activity was doubled when the mitochondria were disrupted; this indicates that the enzyme is localized in the matrix compartment.     

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.     

Carnitine acyltransferase and acyl-coenzyme A hydrolase activities in human liver. Quantitative analysis of their subcellular localization.

The subcellular localizations of carnitine acyltransferase and acyl-CoA hydrolase activities with different chain-length substrates were quantitatively evaluated in human liver by fractionation of total homogenates in metrizamide density gradients and by differential centrifugation. Peroxisomes were found to contain 8-37% of the liver acyltransferase activity, the relative amount depending on the chain length of the substrate. The remaining activity was ascribed to mitochondria, except for carnitine octanoyltransferase, for which 25% of the activity was present in microsomal fractions. In contrast with rat liver, where the activity in peroxisomes is very low or absent, human liver peroxisomes contain about 20% of the carnitine palmitoyltransferase. Short-chain acyl-CoA hydrolase activity was found to be localized mainly in the mitochondrial and soluble compartments, whereas the long-chain activity was present in both microsomal fractions and the soluble compartment. Particle-bound acyl-CoA hydrolase activity for medium-chain substrates exhibited an intermediate distribution, in mitochondria and microsomal fractions, with 30-40% of the activity in the soluble fraction. No acyl-CoA hydrolase activity appears to be present in human liver peroxisomes.     

Deacylation of acetyl-coenzyme A and acetylcarnitine by liver preparations.

The breakdown of acetylcarnitine catalysed by extracts of rat and sheep liver was completely abolished by Sephadex G-25 gel filtration, whereas the hydrolysis of acetyl-CoA was unaffected. Acetyl-CoA and CoA acted catalytically in restoring the ability of Sephadex-treated extracts to break down acetylcarnitine, which was therefore not due to an acetylcarnitine hydrolase but to the sequential action of carnitine acetyltransferase and acetyl-CoA hydrolase. Some 75% of the acetyl-CoA hydrolase activity of sheep liver was localized in the mitochondrial fraction. Two distinct acetyl-CoA hydrolases were partially purified from extracts of sheep liver mitochondria. Both enzymes hydrolysed other short-chain acyl-CoA compounds and succinyl-CoA (3-carboxypropionyl-CoA), but with one acetyl-CoA was the preferred substrate.     

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.     

Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.     

Carnitine acyltransferases in rat liver peroxisomes

Carnitine acyltransferase activities, as well as acetyl-CoA, octanyl-CoA, and palmityl-CoA hydrolase activities, were assayed in mitochondrial, peroxisomal, and endoplasmic reticulum fractions after isopycnic density sucrose gradient fractionation of rat liver homogenates. Both the forward and reverse assays show that carnitine acetyltransferase and carnitine octanyltransferase are associated with peroxisomes, mitochondria, and endoplasmic reticulum, while carnitine palmityltransferase was detected in mitochondria. Palmityl-CoA and octanyl-CoA hydrolase activities were found in all but the leading edge of the peroxisome peak of the gradient. The palmityl-CoA hydrolase in peroxisomal fractions was due to lysosomal contamination since the activity coincided with the lysosomal marker, acid phosphatase. The substrate specificity for carnitine octanyltransferase activity was maximum with medium-chain-length derivatives (about 20 nmol/ min/mg protein) and decreased as the acyl length increased until very low activity (<1 nmol/min/mg protein) was obtained with palmityl-CoA. When acyltransferases in peroxisomes were assayed by measuring acylcarnitine formation, nearly theoretical amounts of acetylcarnitine and octanylcarnitine were formed, but lesser quantities of 12 and 14 carbon acylcarnitines and very low amounts of palmitylcarnitine were detected. The presence of a broad spectrum of medium-chain and short-chain carnitine acyltransferases in peroxisomes is consistent with a role for carnitine for shuttling short-chain and medium-chain acyl residues out of peroxisomes. Carnitine acyltransferase activity was not detected in peroxisomes from spinach leaves.     

Mitochondrial enzymes responsible for oxidizing medium-chain fatty acids in developing rat skeletal muscle, heart, and liver

Prior to weaning, medium-chain fatty acids constitute an important energy source in the developing rat. Fatty acid oxidation rates increase with age in most developing tissues, but the pattern of this increase may vary according to the role of the particular organ. In skeletal muscle, heart, and liver of developing rats, we measured mitochondrial activities of long- and short-chain enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and long- and short-chain acyl-CoA thiolase. In skeletal muscle, the pattern of development in fatty acid oxidation enzymes favored utilization of long-chain rather than medium-chain fatty acids. In liver, enzyme activities for medium-chain fatty acids were highest prior to weaning. Heart occupied a position intermediate between skeletal muscle and liver.     

Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues.

The effects of Ca2+ on the activity of isocitrate dehydrogenase (NAD+) in extracts of rat heart mitochondria were explored in the presence of MgCl2 by using EGTA buffers. In the absence of ADP, Ca2+ (about 30 micrometer) resulted in a slight increase in apparent Km for threo-Ds-isocitrate; in the presence of ADP, Ca2+ (about 25 micrometer) greatly lowered the apparent Km for threo-Ds-isocitrate from 227 micrometer to 53 micrometer without changing the maximum velocity. At 100 micrometer-threo-Ds-isocitrate and 1 mM-ADP, there was an 8-fold activation by Ca2+, with a Km for Ca2+ of 1.2 micrometer. This activation was also observed with Sr2+ (Km 3.1 micrometer), but not with Mn2+ (at concentrations below 2.5 micrometer). Similar effects of Ca2+ were also observed on isocitrate dehydrogenase (NAD+) activity in extracts of mitochondria from liver, kidney, brown adipose tissue and white adipose tissue of the rat. The possible regulatory role of changes in the intramitochondrial concentration of Ca2+ is discussed.     

Acyl-CoA dehydrogenase activity in pea cotyledon tissue during germination and initial growth

Acyl-CoA dehydrogenase activity has been measured in homogenates of post-imbibition to 14-day-old hydroponically grown pea seeds at daily intervals, using C(4), C(12) and C(16) acyl-CoA substrates. The activity peaks of the different chain-length acyl-CoA dehydrogenases did not transpose at all points and the ratios of the chain-length activities were not constant. It therefore has to be concluded that more than one dehydrogenase is present in pea mitochondria. There was a post-imbibition initial surge of activity with short- and mid-chain-length substrates. The C(16)-handling enzyme first peaked at 3-4 days, which coincided with the onset of plumule unfurling and greening. Further peaks were observed with all three substrates, coinciding with secondary root formation and leaf enlargement and later with cotyledon degeneration. Overall activity showed that the long-chain acyl-CoA dehydrogenase was much more active than the short-chain acyl-CoA dehydrogenase.     

On the mechanism of malonyl-CoA-independent fatty-acid synthesis. Different properties of the mitochondrial chain elongation and enoylCoA reductase in various tissues.

1. NADPH-specific mitochondrial enoyl-CoA reductase can be assayed by a sensitive radioactive test, employing tritium-labelled NADPH, synthesized in a prefixed reaction from D-[1-3H]-glucose via the hexokinase and glucose-6-phosphate dehydrogenase reactions. 2. Liver, kidney cortex, heart muscle, skeletal muscle, brown adipose tissue, brain cortex, and aortic intimal tissue are investigated concerning chain lengths specificity of the chain elongation and the enoyl-CoA reductase. Medium-chain acyl-CoA compounds prove to be the best primers for the chain elongation. Enoyl-CoA reductases still show large incorporation rates with hexadecenoyl-CoA. 3. The differences in the chain lengths specificity of the chain elongation and enoyl-CoA reductase can be explained by the inhibitory effect of long-chain acyl-CoA derivatives on the 3-hydroxyacyl-CoA dehydrogenase. 4. The nucleotide specificity in the different tissues reveals two types of chain elongation: In addition to liver and kidney cortex, mitochondria of brown adipose tissue need NADH + NADPH for optimal chain elongation, whereas heart muscle, skeletal muscle and aortic intimal mitochondria only need NADH. 5. Different physiological roles are proposed for the two types. The "heart type" may be of importance in the conservation of reducing equivalents or acetate units in the anaerobic state, the "liver type" may play a role in the transfer of hydrogen from NADPH to the respiratory chain. In addition, the mitochondrial chain elongation may serve as bypass of the first part of the respiratory chain.     

Specific properties of brown adipose tissue mitochondrial membrane.

1. Mitochondrial membrane of brown adipose tissue compared to that of liver possesses a very high activity of oxidative enzymes but a low activity of ATPase. 2. The polypeptide composition of the mitochondrial membranes proves that the above differences in enzyme activities are due to increased content of oxidative enzymes and decreased content of ATPase in brown adipose tissue. 3. The inhibition of ATPase of brown adipose tissue mitochondria by aurovertin, oligomycin and DCCD indicates modified proportions between the components of the ATPase complex. 4. The organization of brown adipose tissue mitochondrial membrane in relation to its thermogenic function is discussed.     

Sex-related difference in the effect of clofibric acid on induction of two novel long-chain acyl-CoA hydrolases in rat liver

The sex differences in the induction of two novel long-chain acyl-CoA hydrolases in hepatic cytosol of rats by clofibric acid (p-chlorophenoxyisobutyric acid)-feeding and the properties of the induced acyl-CoA hydrolases were investigated. Marked sex-related difference was observed in the induction of acyl-Coa hydrolase activity. The sex difference was mainly due to the difference in the induction of acyl-CoA hydrolase with higher molecular weight (hydrolase I), but not to the difference in the induction of acyl-CoA hydrolase with lower molecular weight (hydrolase II). The extent of the induction of the hydrolase I in hepatic cytosol of male rats was 3.5 times over that of female rats. Castration of male rats resulted in the marked depression of the ability to induce hydrolase I. The administration of testosterone to the castrated male rats recovered completely the ability to induce hydrolase I. Unlike hydrolase I, the ability to induce hydrolase II did not respond to the changes in state of androgen. The administration of di-(2-ethylhexyl)phthalate also induced both hydrolase I and II, although the extent of the induction of hydrolase I was less compared to that by clofibric acid treatment. Likewise, marked sex difference was observed in the induction of the hydrolase I on di-(2-ethylhexyl)phthalate administration. These two hydrolases showed different kinetic properties and different substrate specificities to each other. Hydrolase I was inhibited by bovine serum albumin in vitro, but was not affected by Mg2+. Hydrolase II was activated slightly in the presence of lower concentrations of bovine serum albumin, Mg2+ or Ca2+.     

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

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

Calcium sensitive isocitrate and 2-oxoglutarate dehydrogenase activities in rat liver and AS-30D hepatoma mitochondria

NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase in extracts of mitochondria from the highly malignant AS-30D rat hepatoma cell line demonstrate Ca2+ sensitivities and affinities for substrates similar to those of normal liver mitochondria. However, the maximal activities of NAD+- and NADP+-dependent isocitrate dehydrogenase were found to be 8 and 3.5 fold higher in hepatoma mitochondrial extracts than those of liver mitochondria, whereas maximal activities of succinate and 2-oxoglutarate dehydrogenases were similar in the two tissues. At pyridine nucleotide concentrations giving the lowest physiological NADH/NAD+ ratio, NAD+-isocitrate dehydrogenase activity in hepatoma mitochondrial extracts was completely inhibited at subsaturating concentrations of Ca2+, substrate, and NAD+, in contrast to rat liver mitochondrial extracts which retained significant activity.     

Developmental changes in the characteristics of peroxisomal fatty acid oxidation system in rat liver

Developmental changes in fatty acid oxidation system of rat liver peroxisomes were studied to compare with that of mitochondria. More apparent enhancement of peroxisomal palmitoyl-CoA oxidase was observed than mitochondrial palmitoyl-CoA dehydrogenase during prenatal (20-day fetal) to neonatal (1-day after birth) period. The characteristics of peroxisomal enzymes, fatty acyl-CoA oxidase and carnitime acyltransferase, on the bases of substrate specificities, were rapidly established within the 1 day after birth accompanied by the marked enhancement of these activities. These findings indicate that peroxisomal fatty acid oxidation system plays an important role for early growth of neonatal rats; this system may contribute to supplying short- to medium-chain fatty acyl-CoA and NADH₂ for mitochondrial energy formation system.     

Expression and purification of His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase wild-type and Arg256 mutant proteins

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. We cloned the gene of rat mitochondrial medium-chain acyl-CoA dehydrogenase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 3' of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 88% yield to apparent homogeneity. The specific activity of the purified His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase was 4.0 U/mg. Arg256 is a highly conserved amino acid, which may play an important role in enzymatic reaction based on the crystal structure of medium-chain acyl-CoA dehydrogenase. We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Arg256 is a very important residue of rat mitochondrial medium-chain acyl-CoA dehydrogenase. Our overexpression in E. coli and one-step purification of the highly active rat mitochondrial medium-chain acyl-CoA dehydrogenase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of medium-chain acyl-CoA dehydrogenase.