Effect of clofibrate treatment on carnitine acyltransferases in different subcellular fractions of rat liver.

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyl- transferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fractionation. In normal liver 48% of total carnitine acetyltransferase activity was peroxisomal, 36% of the activity located in mitochondria and 16% in a membranous fraction containing microsomes. Carnitine octanoyltransferase and carnitine palmitoyltransferase were confined almost totally (77--81%) to mitochondria in normal liver. Clofibrate treatment increased the total activity of carnitine acetyltransferase over 30 times, whereas the total activities of the other two transferases were increased only 5-fold. From the three different subcellular carnitine acetyltransferases the mitochondrial one was most responsive to clofibrate treatment, i.e. the rise in mitochondrial activity was over 70-fold as contrasted to the 6- and 14-fold rises in peroxisomal and microsomal activities, respectively. After treatment mitochondria contained 79% of total activity. It is concluded that the clofibrate-induced increase of carnitine acetyltransferase activity is not due to the peroxisomal proliferation that occurs during clofibrate treatment. The rise in peroxisomal activity contributed only 8% to the total increase. After clofibrate treatment the greatest part of carnitine octanoyl- and palmitoyltransferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.     

Effect of clofibrate on peroxisomal and mitochondrial beta-oxidation in chicken liver

The effect of a 0.25% clofibrate diet for 2 weeks on peroxisomal and mitochondrial beta-oxidation in chicken liver was studied. The activities of antimycin antimycin A-insensitive palmitoyl-CoA oxidation (peroxisomal beta-oxidation) and carnitine acetyltransferase increased about two-fold. The activities of palmitoyl-CoA-dependent O2 consumption (mitochondrial beta-oxidation) and carnitine palmitoyltransferase were also slightly activated by the administration of clofibrate, but not significant. Thus, clofibrate may be a typical drug which activates the peroxisomal beta-oxidation more than the mitochondrial one in various species. The effect of clofibrate on peroxisomal carnitine acetyltransferase was the same as that on the mitochondrial one in chicken liver. Serum lipids were not lowered, but hepatomegaly was observed in the present experiment with chicken.     

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.     

Carnitine octanoyltransferase of mouse liver peroxisomes: properties and effect of hypolipidemic drugs

Carnitine octanoyltransferase (COT) in 500g supernatant fluids from mouse liver has a specific activity at least twice that of carnitine acetyltransferase (CAT) or carnitine palmitoyltransferase (CPT). When mice are fed diets containing the lipid-lowering drugs, clofibrate or nafenopin, the specific activity of COT increases 4- and 11-fold, respectively. Liver homogenates from mice fed a control diet, and diets containing clofibrate, nafenopin, or Wy-14,643 were fractionated by sucrose gradient centrifugation, and the subcellular distribution of carnitine acyltransferases was determined. In the controls, peroxisomes contained about 70% of the total COT. The specific activity of COT in the peroxisomal peak was 12-fold greater than either CAT or CPT, and 20-fold greater than the COT activity in the mitochondrial fraction. Treatment with hypolipidemic drugs increased the specific activity of peroxisomal COT 2- to 3-fold and CAT 6- to 12-fold, while mitochondrial COT increased 5- to 11-fold and CAT 19- to 54-fold. COT was purified to homogeneity from livers of mice treated with Wy-14,643. It had an apparent Mr of 60,000 by Sephadex G-100 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a maximum activity for octanoyl-CoA with acetyl-CoA and palmitoyl-CoA having activities of 2 and 10%, respectively, when 100 microM acyl-CoA substrates were used. The Km's for 1-carnitine, octanoyl-CoA, palmitoyl-CoA, and acetyl-CoA were 130, 15, 69, and 155 microM, respectively, in the forward direction; and in the reverse direction were 110, 100, 104, and 783 microM for CoASH, octanoylcarnitine, palmitoylcarnitine, and acetylcarnitine, respectively. With Vmax conditions, acetyl-CoA and palmitoyl-CoA had activities of 8 and 26% of the activity for octanoyl-CoA, and acetylcarnitine and palmitoylcarnitine had activities of 7 and 22%, respectively, of the activity for octanoylcarnitine. It is concluded that COT is a separate enzyme present in large amounts in the matrix of mouse liver peroxisomes, with kinetic properties that greatly favor medium-chain acylcarnitine formation.     

Characteristics of the suppressive effect of nicardipine on peroxisome induction in rat liver

In vivo administration of nicardipine, a known calcium antagonist, suppressed the clofibrate-evoked induction of activities of peroxisomal enzymes, such as catalase, the peroxisomal fatty acyl-CoA oxidizing system, carnitine acetyltransferase and mitochondrial carnitine palmitoyltransferase in rat liver. On a time-course study, the suppression of induction in the activities of the peroxisomal fatty acyl-CoA oxidizing system and carnitine acetyltransferase was found at 5 days after the treatment, whereas the induction by clofibrate was already observed at 1 day after the treatment, suggesting that in the process of peroxisome induction by clofibrate there might be two steps, i.e., a triggering step and an enhancing step, and nicardipine might act as suppressor for the later step. The precursor-incorporation studies with [3H]leucine showed that the rate of the synthesis of the peroxisomal bifunctional enzyme was increased by 4.2-fold after clofibrate-treatment, whereas nicardipine suppressed this enhancement to only 2.2-fold of the control. The rate of degradation of this enzyme was not affected by any treatment. These results show that nicardipine affects the regulation mechanism of the biosynthesis of this enzyme. Nicardipine showed hardly any suppressive-effect on the hepatic peroxisomal enzyme induction observed in high-fat diet fed rat. Furthermore, the suppression of clofibrate-evoked induction of peroxisomal enzymes was observed also in mice. These interesting findings suggest that there is a difference in the mechanism of peroxisome proliferation and/or the induction of peroxisomal enzymes between clofibrate and physiological conditions, such as high-fat diet feeding. The suppression of drug-induced peroxisome proliferation by calcium antagonists may help in dissecting the causal relationship between the multiple effects mediated by peroxisomal proliferators.     

Enzymes of carnitine acylation. Is overt carnitine palmitoyltransferase of liver peroxisomal carnitine octanoyltransferase?

Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.     

Purification and properties of peroxisomal carnitine palmitoyltransferase in chick embryo liver

Peroxisomal carnitine palmitoyltransferase was purified by solubilization using Tween 20 and KCl from the large granule fraction of the liver of clofibrate-treated chick embryo, DEAE-Sephacel and blue Sepharose CL-6B column chromatography. The peroxisomal carnitine palmitoyltransferase was an Mr 64,000 polypeptide; the mitochondrial carnitine palmitoyltransferase had a subunit molecular weight of 69,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carnitine acetyltransferase was an Mr 64,000 polypeptide. Antibody against purified peroxisomal carnitine palmitoyltransferase reacted only with peroxisomal carnitine palmitoyltransferase, but not with mitochondrial carnitine palmitoyltransferase or carnitine acetyltransferase. In addition, anti-peroxisomal carnitine palmitoyltransferase reacted only with the protein in peroxisomes purified from chick embryo liver by sucrose density gradient centrifugation. Thus, it was confirmed that purified peroxisomal carnitine palmitoyltransferase was a peroxisomal protein. Compared with mitochondrial carnitine palmitoyltransferase, peroxisomal carnitine palmitoyltransferase was extremely resistant to inactivation by trypsin. The pH optimum of peroxisomal carnitine palmitoyltransferase was 8.5, differing from that of mitochondrial carnitine palmitoyltransferase. The Km value of peroxisomal carnitine palmitoyltransferase for palmitoyl-CoA (32 microM) was similar to that of the mitochondrial one, whereas those values for L-carnitine (140 microM), palmitoyl-L-carnitine (43 microM) and CoA (9 microM) were lower than those of mitochondrial carnitine palmitoyltransferase. Peroxisomal carnitine palmitoyltransferase exhibited similar substrate specificities in both the forward and reverse reactions, with the highest activity toward lauroyl derivatives. Furthermore, this enzyme showed relatively high affinities for long-chain acyl derivatives (C10-C16) and similar Km values (30-50 microM) for acyl-CoAs, acylcarnitine and CoA, and a constant Km value (approximately 150 microM) for carnitine. These results indicate that peroxisomal carnitine palmitoyltransferase played a role in the modulation of the intracellular CoA/long-chain acyl-CoA ratio at the hatching stage of chicken when long-chain fatty acids are actively oxidized in peroxisomes.     

Clofibrate induces carnitine acyltransferases in periportal and perivenous zones of rat liver and does not disturb the acinar zonation of gluconeogenesis

Clofibrate induces hypertrophy and hyperplasia and marked changes in the activities of various enzymes in rat liver. We examined the effects of treatment of rats with clofibrate on enzyme induction and on rates of metabolic flux in hepatocytes isolated from the periportal and perivenous zones of the liver. Clofibrate induced the activities of carnitine acetyltransferase (90-fold), carnitine palmitoyltransferase (3-fold) and NADP-linked malic enzyme (3-fold) to the same level in periportal as in perivenous hepatocytes, suggesting that these enzymes were induced uniformly throughout the liver acinus. Increased rates of palmitate metabolism and ketogenesis after clofibrate treatment were associated with: a more oxidised mitochondrial redox state; diminished responsiveness to glucagon and loss of periportal/perivenous zonation. Despite the marked liver enlargement and hyperplasia caused by clofibrate, the normal periportal/perivenous zonation of alanine aminotransferase and gluconeogenesis was preserved in livers of clofibrate-treated rats, indicating that clofibrate-induced hyperplasia does not disrupt the normal acinar zonation of these metabolic functions.     

Involvement of carnitine acyltransferases in peroxisomal fatty acid metabolism by the yeast Pichia guilliermondii.

This article provides information about peroxisomal fatty acid metabolism in the yeast Pichia guilliermondii. The existence of inducible mitochondrial carnitine palmitoyltransferase and peroxisomal carnitine octanoyl-transferase activities was demonstrated after culture of this yeast in a medium containing methyl oleate. The subcellular sites and induction patterns were studied. The inhibition of carnitine octanoyl- and palmitoyl-transferases by chlorpromazine to a large extent prevented the otherwise observed metabolism-dependent inactivation of thiolase by 2-bromofatty acids in vivo. We concluded that the metabolism of long- and medium-chain fatty acids in the peroxisome of this yeast involved carnitine intermediates.     

Effect of clofibrate application on morphology and enzyme content of liver peroxisomes.

Male albino rats (Sprague Dawley) were fed for 2-6 weeks on a diet containing 0.75% clofibrate. Liver cell fractions obtained from these animals were assayed for peroxisomal enzymes. In the cell homogenate the catalase activity was doubled, whereas the activity of urate oxidase was found to be only slightly depressed. The activity of carnitine acetyltransferase increased several times. In liver peroxisomes purified by isopycnic gradient centrifugation the specific activity of urate oxidase decreased appreciably showing that peroxisomes formed under the proliferative influence of clofibrate are not only modified with respect to their morphological characteristics but also to their enzymic equipment. This is also obvious from the changes in peroxisomal carnitine acetyltransferase activity which was enhanced by clofibrate to more than the fivefold amount. In purified mitochondria this enzyme was even more active: clofibrate advances both, the peroxisomal and the mitochondrial moiety of carnitine acetyltransferase. Morphological and cytochemical studies showed an increase in the number of microbodies and as compared to the controls microbodies were lying in groups more frequently. Small particles located closely adjacent to "normal" sized peroxisomes were found particularly after short feeding periods. While the number of coreless microbodies increased studies gave no clear evidence for an increase in marked shape irregularities of the peroxisomes.     

Study of some factors controlling fatty acid oxidation in liver mitochondria of obese Zucker rats.

Livers of genetically obese Zucker rats showed, compared with lean controls, hypertrophy and enrichment in triacylglycerols, indicating that fatty acid metabolism was directed towards lipogenesis and esterification rather than towards fatty acid oxidation. Mitochondrial activities of cytochrome c oxidase and monoamine oxidase were significantly lower when expressed per g wet wt. of liver, whereas peroxisomal activities of urate oxidase and palmitoyl-CoA-dependent NAD+ reduction were unchanged. Liver mitochondria were able to oxidize oleic acid at the same rate in both obese and lean rats. For reactions occurring inside the mitochondria, e.g. octanoate oxidation and palmitoyl-CoA dehydrogenase, no difference was found between both phenotypes. Total carnitine palmitoyl-, octanoyl- and acetyl-transferase activities were slightly higher in mitochondria from obese rats, whereas the carnitine content of both liver tissue and mitochondria was significantly lower in obese rats compared with their lean littermates. The carnitine palmitoyltransferase I activity was slightly higher in liver mitochondria from obese rats, but this enzyme was more sensitive to malonyl-CoA inhibition in obese than in lean rats. The above results strongly suggest that the impaired fatty acid oxidation observed in the whole liver of obese rats is due to the diminished transport of fatty acids across the mitochondrial inner membrane via the carnitine palmitoyltransferase I. This effect could be reinforced by the decreased mitochondrial content per g wet wt. of liver. The depressed fatty acid oxidation may explain in part the lipid infiltration of liver observed in obese Zucker rats.     

Characterization of peroxisomes in Tetrahymena pyriformis

Peroxisomes from Tetrahymena pyriformis contained catalase, d-amino acid oxidase, cyanide-insensitive fatty acyl-CoA oxidizing system, carnitine acetyltransferase, isocitrate lyase, leucine:glyoxylate aminotransferase and phenylalanine:glyoxylate aminotransferase. These activities, except carnitine acetyltransferase, were found at the highest levels in the light mitochondrial fraction, whereas the highest activity of carnitine acetyltransferase was found in the micotchondrial fraction. Sucrose density gradient centrifugation showed that the density of peroxisomes was approx. 1.228 g/ml and that of mitochondria was approx. 1.213 g/ml. When the light mitochondrial fraction was treated with deoxycholate or by freeze-thawing, most of the activities of catalase and isocitrate lyase were solubilized, whereas about half of the original activity of aminotransferase remained in the pellet fraction. Addition of fatty acid and clofibrate increased the activities of the cyanide-insensitive fatty acyl-CoA oxidizing system and isocitrate lyase in the peroxisomes. The activity of catalase was slightly increased by glucose and clofibrate; leucine:glyoxylate aminotransferase activity was significantly increased by clofibrate treatment.     

Correlation between the cellular level of long-chain acyl-CoA, peroxisomal beta-oxidation, and palmitoyl-CoA hydrolase activity in rat liver. Are the two enzyme systems regulated by a substrate-induced mechanism?

Data obtained in earlier studies with rats fed diets containing high doses of peroxisome proliferators (niadenate, tiadenol, clofibrate, or nitotinic acid) are used to look for a quantitative relationship between peroxisomal beta-oxidation, palmitoyl-CoA hydrolase, palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities, and the cellular concentration of their substrate and reaction products. The order of the hyperlipidemic drugs with regard to their effect on CoA derivatives and enzyme activities was niadenate greater than tiadenol greater than clofibrate greater than nicotinic acid. Linear regression analysis of long-chain acyl-CoA content versus palmitoyl-CoA hydrolase and peroxisomal beta-oxidation activity showed highly significant linear correlations both in the total liver homogenate and in the peroxisome-enriched fractions. A dose-response curve of tiadenol showed that carnitine palmitoyltransferase and palmitoyl-CoA synthetase activities and the ratio of long-chain acyl-CoA to free CoASH in total homogenate rose at low doses before detectable changes occurred in the peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. A plot of this ratio parallelled the palmitoyl-CoA synthetase activity. The specific activity of microsomally localized carnitine palmitoyl-transferase was low and unchanged up to a dose where no enhanced peroxisomal beta-oxidation was observed, but over this dose the activity increased considerably so that the specific of the enzyme in the mitochondrial and microsomal fractions became comparable. The mitochondrial palmitoyl-CoA synthetase activity decreased gradually. The correlations may be interpreted as reflecting a common regulation mechanism for palmitoyl-CoA hydrolase and peroxisomal beta-oxidation enzymes, i.e., the cellular level of long-chain acyl-CoA acting as the metabolic message for peroxisomal proliferation resulting in induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase activity. The findings are discussed with regard to their possible consequences for mitochondrial fatty acid oxidation and the conversion of long-chain acyl-L-carnitine to acyl-CoA derivatives.     

Physiological role of peroxisomal beta-oxidation in liver of fasted rats

In the livers of fasted rats, the activity of peroxisomal palmitocyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial beta-oxidation. The peroxisomal oxidizing activity was about twice that of the control throughout the period of fasting (1-7 days). carnitine acetyltransferase activity was increased to a similar extent in both peroxisomes and mitochondria. A possible physiological role of liver peroxisomes may thus be as an effective supply of NADH2, acetyl residues and short and medium-length fatty acyl-CoA in the cells on the enhancement of peroxisomal beta-oxidation of the animals under starvation; these substances thus produced may be transported into the mitochondria as energy sources.     

Developmental changes in the activities of peroxisomal and mitochondrial beta-oxidation in chicken liver

The activities of peroxisomal and mitochondrial beta-oxidation and carnitine acyltransferases changed during the process of development from embryo to adult chicken, and the highest activities of peroxisomal beta-oxidation, palmitoyl-CoA oxidase, and carnitine acetyltransferase were found at the hatching stage of the embryo. The profiles of these alterations were in agreement with those of the contents of triglycerides and free fatty acids in the liver. The highest activities of mitochondrial beta-oxidation and palmitoyl-CoA dehydrogenase were observed at the earlier stages of the embryo; then the activities decreased gradually from embryo to adult chicken. The ratio of activities of carnitine acetyltransferase in peroxisomes and mitochondria (peroxisomes/mitochondria) increased from 0.54 to 0.82 during the development from embryo to adult chicken. The ratio of activities of carnitine palmitoyltransferase decreased from 0.82 to 0.25 during the development. The affinity of fatty acyl-CoA dehydrogenase toward the medium-chain acyl-CoAs (C6 and C8) was high in the embryo and decreased with development, whereas the substrate specificity of fatty acyl-CoA oxidase did not change. The substrate specificity of mitochondrial carnitine acyltransferases did not change with development. The affinity of peroxisomal carnitine acyltransferases toward the long-chain acyl-CoAs (C10 to C16) was high in the embryo, but low in adult chicken.     

Effects of fat content in the diet on hepatic peroxisomes of the rat

Effects of fat content in the diet on rat liver peroxisomes was examined. In the livers of rats fed for one week on the high-fat diet containing 30% fat, the cyanide-insensitive palmitoyl-CoA oxidation was accelerated to eight times that of control and the enzymic activities of catalase, carnitine acetyltransferase and carnitine palmitoyltransferase were elevated by the factors of 1.3, 5 and 2, respectively. In contrast, the activities of D-amino acid oxidase in addition to the three enzymes mentioned above were all lowered by 20% when the animals were maintained on a fat-free diet for the same period of time. It appears that the high-fat diet-induced increase in the activity of carnitine palmitoyltransferase is a result of the raised activity of this enzyme in mitochondria only while the apparent high activity reflects stimulation of carnitine acetyltransferase in all the subcellular fractions. Another notable effect of the high-fat diet was a remarkable increase in the quantity of a peroxisome-associated polypeptide which was separable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is noteworthy that this effect of the high-fat diet resemble that of clofibrate. If the diet was deprived of fat, however, this polypeptide species, with an estimated molecular weight of 80 000, decreased to a level slightly lower than normal. On the basis of the electron micrographic criteria, the high-fat diet provoked a marked proliferation of hepatic peroxisomes.     

Detection of heat-stable delta 3,delta 2-enoyl-CoA isomerase in rat liver mitochondria and peroxisomes by immunochemical study using specific antibody

The subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the peroxisomal isomerase, revealing a single band with an apparent molecular weight of 30,000. However, the isomerase was induced by clofibrate administration mainly in the mitochondrial fraction. These results suggest that delta 3,delta 2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.     

Purification of heart and liver mitochondrial carnitine acetvltransferase

Heart and liver mitochondrial, as well as liver peroxisomal, carnitine acetyltransferase was purified to apparent homogeneity and some properties, primarily of heart mitochondrial carnitine acetyltransferase, were determined. Hill coefficients for propionyl-CoA are 1.0 for each of the enzymes. The molecular weight of heart mitochondrial carnitine acetyltransferase, determined by SDS-PAGE, is 62,000. It is monomeric in the presence of catalytic amounts of substrate. Polyclonal antibodies against purified rat liver peroxisomal carnitine acetyltransferase precipitate liver and heart mitochondrial and liver peroxisomal carnitine acetyltransferase, but not liver peroxisomal carnitine octanoyltransferase. Liver peroxisomes, mitochondria, and microsomes and heart mitochondria all give multiple bands on Western blotting with the antibody against carnitine acetyltransferase. Major protein bands occur at the molecular weight of carnitine acetyltransferase and at 33 to 35 kDa.     

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.     

Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.