Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase in rat liver. The effects of di-(2-ethylhexyl)phthalate, a peroxisome proliferator

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase (isomerase) was investigated in rat liver. Livers of di-(2-ethylhexyl)phthalate (DEHP)-treated or untreated rats were perfusion-fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus-lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus-lining cells decreased. The isomerase staining pattern was quite similar to that of long-chain acyl-CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus-lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2-fold whereas the mitochondrial isomerase was enhanced about 5-fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.     

Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities

Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.     

Occurrence of a long-chain delta 3,delta 2-enoyl-CoA isomerase in rat liver.

Isoproteins of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8), an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids having double bonds at odd-numbered positions, were studied in livers of control and clofibrate-treated rats. When liver extracts were applied to a hydroxyapatite column at pH 7.0, the previously characterized peroxisomal trifunctional hydratase-dehydrogenase-isomerase enzyme and the mitochondrial isomerase, which shows a preference for short-chain substrates, were eluted almost in parallel. In addition to these activities, a separate isomerase was observed to elute at a lower potassium phosphate concentration in the gradient. Experiments with extracts of purified mitochondria and peroxisomes demonstrated the mitochondrial origin of this third activity. Studies on the kinetic properties of the third isomerase showed that it has a preference for C10-C12 substrates. An Mr of 200,000 was obtained for the native protein by gel-filtration chromatography. Antibodies to mitochondrial short-chain isomerase and peroxisomal trifunctional enzyme did not recognize this novel mitochondrial isoenzyme. The immunological non-cross-reactivity can be interpreted as suggesting that the different isomerases are not closely related at the level of the primary structure of the polypeptide chain. The present data demonstrate that, similar to many other enzymes of beta-oxidation, delta 3,delta 2-enoyl-CoA isomerase has at least three isoenzymes in rat liver: mitochondrial short- and long-chain isomerases and an additional peroxisomal isoenzyme, which in this case is a part of a multifunctional protein.     

Beta-oxidation of polyunsaturated fatty acids in peroxisomes. Subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase activity in rat liver

The metabolism of the double bonds at the delta 3 position in fatty acids was studied in rat liver. Infusion of delta 3-trans-dodecenoic acid into isolated perfused liver and subcellular fractionation studies showed the presence of both peroxisomal and mitochondrial delta 3,delta 2-enoyl-CoA isomerase activity (EC 5.3.3.8). These findings together with the previous demonstration of peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34) [(1981) J. Biol. Chem. 256, 8259-8262] and D-3-OH-acyl-CoA epimerase (EC 5.1.2.3) [(1985) FEBS Lett. 185, 129-134] activities show that peroxisomes possess all the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids.     

Delta 3,delta 2-enoyl-CoA isomerases. Characterization of the mitochondrial isoenzyme in the rat

Delta 3,delta 2-Enoyl-CoA isomerase (EC 5.3.3.8), an obligatory auxiliary enzyme for the metabolism of double bonds at odd-numbered positions of fatty acids during their beta-oxidation, was studied in hearts and livers of normal and clofibrate-treated rats. Hepatic peroxisomal and mitochondrial isoenzymes were separable by dye-ligand chromatography. The mitochondrial one was further purified to apparent homogeneity. An isomerase was also purified from heart muscle, a peroxisome-poor tissue. These enzymes were dimeric basic proteins (pI 9.5) with a subunit molecular weight of 30,000. Both cis- and trans-enoyl-CoA served as substrates for the hepatic enzyme studied. The velocity ratio for the C6-, C10-, and C12-trans-3-enoyl substrates was 9:2.5:1. By immunoelectron microscopy the enzyme protein selected for purification was found to be mitochondrial both in liver and heart. Chromatographic evidence, immunoelectron microscopy, and immunoblotting indicated that in the liver but not in the heart, the enzyme underwent an induction of 1 order of magnitude during clofibrate treatment. Antibodies towards the rat isomerases detected cross-reactive proteins in bovine and pig liver and heart and human placenta. The estimated subunit sizes varied from species to species, being 31,000 in bovine liver and heart, 29,000 in pig liver and heart, and 30,000 in human placenta. The data are in accord with the notion of a dual location of the delta 3,delta 2-enoyl-CoA isomerase. Mitochondrial origin of one of the isoenzymes and its tissue-specific induction by clofibrate were verified by immunochemistry and the identity of the peroxisomal one revealed by the chromatographic behavior of the proteins.     

Investigation of the presence of branched-chain alpha-keto acid dehydrogenase in mammalian hepatic peroxisomes.

1. Rat liver was fractionated into peroxisomes and mitochondria and branched-chain keto acid (BCKA) dehydrogenase activity was measured. 2. All BCKA dehydrogenase activity was associated with the mitochondrial fraction and none with the peroxisomal fraction. 3. BCKA dehydrogenase was also not detected in hepatic peroxisomes of rats treated with clofibrate which induces several peroxisomal enzymes. 4. Hepatic peroxisomes from rabbit, hamster and dog also did not show any BCKA dehydrogenase activity. 5. We conclude that mammalian hepatic peroxisomes do not contain BCKA dehydrogenase.     

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.     

Hepatic enzymes, CoASH and long-chain acyl-CoA in subcellular fractions as affected by drugs inducing peroxisomes and smooth endoplasmic reticulum

1. The activities of acyl-CoA hydrolase, catalase, urate oxidase and peroxisomal palmitoyl-CoA oxidation as well as the protein content and the level of CoASH and long-chain acyl-CoA were measured in subcellular fractions of liver from rats fed diets containing phenobarbital (0.1% w/w) or clofibrate (0.3% w/w). 2. Whereas phenobarbital administration resulted in increased microsomal protein, the clofibrate-induced increase was almost entirely attributed to the mitochondrial fraction with minor contribution from the light mitochondrial fraction. 3. The specific activity of palmitoyl-CoA hydrolase in the microsomal fraction was only slightly affected while the mitochondrial enzyme was increased to a marked extent (3-4-fold) by clofibrate. 4. Phenobarbital administration mainly enhanced the microsomal palmitoyl-CoA hydrolase. 5. The increased long-chain acyl-CoA and CoASH level observed after clofibrate treatment was mainly associated with the mitochondrial, light mitochondrial and cytosolic fractions, while the slight increase in the levels of these compounds found after phenobarbital feeding was largely of microsomal origin. 6. The findings suggest that there is an intraperoxisomal CoASH and long-chain acyl-CoA pool. 7. The specific activity of palmitoyl-CoA hydrolase, catalase and peroxisomal palmitoyl-CoA oxidation was increased in the lipid-rich floating layer of the cytosol-fraction. 8. The changes distribution of the peroxisomal marker enzymes and microsomal palmitoyl-CoA hydrolase after treatment with hypolipidemic drugs may be related to the origin of peroxisomes.     

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.     

The effect of clofibrate on amphibian hepatic peroxisomes.

Liver peroxisomes of two anuran amphibian species, Rana esculenta and Xenopus laevis, were studied in untreated and in clofibrate-treated adults by means of complementary technical approaches, ie, ultrastructural cytochemistry, cell fractionation and marker enzyme activity assays. In untreated adults, hepatic peroxisomes were found to be very scarce in Xenopus when compared to Rana. Activities of catalase, D-amino acid oxidase and of the three first enzymes of the peroxisomal beta-oxidation system were detected in the light mitochondrial fractions enriched in peroxisomes and prepared from livers of both species. Administration of clofibrate at a daily dose level of 60 mg (Rana) and 90 mg (Xenopus) during ten days induced a drastic peroxisome proliferation in Rana hepatocytes but had no visible effect on the hepatic peroxisomal population of Xenopus. The catalase activity and the peroxisomal beta-oxidation system of liver cells were enhanced in Rana as well as in Xenopus. The hepatic D-amino acid oxidase specific activity was increased in Rana whereas it remained rather constant in Xenopus. Taking advantage of the behaviors of Rana and Xenopus hepatic peroxisomes, the molecular mechanisms of clofibrate induction are now investigated in the target liver cells of the two amphibian species.     

Effect of clofibrate on intracellular enzyme distribution in the rat liver

The effect of chronic administration of a hypolipaemic agent--clofibrate--on the subcellular distribution of liver enzymes in male rats was studied. Clofibrate produced an increase in the number of peroxisomes and also enhanced the activity of aconitase and histidine: glyoxylate aminotransferase (HGA) in liver homogenate. Differential centrifugation of homogenate revealed an elevation of the relative amounts of catalase, HGA and isocitrate dehydrogenase in the soluble cell fraction in clofibrate pretreated animals. Clofibrate induced peroxisomal HGA but failed to alter the amounts of catalase, urate oxidase and isocitrate dehydrogenase in the particles. In both the experimental and control groups the activity of aconitase, malate dehydrogenase (NAD+), creatine phosphokinase and glutathione reductase was observed in mitochondrial fractions and was not detected in purified peroxisomes.     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of 'small peroxisomes', containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.     

Functional characterization of Delta3,Delta2-enoyl-CoA isomerases from rat liver.

The degradation of unsaturated fatty acids by beta-oxidation involves Delta(3),Delta(2)-enoyl-CoA isomerases (enoyl-CoA isomerases) that catalyze 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of enoyl-CoAs and the 2,5 --> 3,5 isomerization of dienoyl-CoAs. An analysis of rat liver enoyl-CoA isomerases revealed the presence of a monofunctional enoyl-CoA isomerase (ECI) in addition to mitochondrial enoyl-CoA isomerase (MECI) in mitochondria, whereas peroxisomes contain ECI and multifunctional enzyme 1 (MFE1). Thus ECI, which previously had been described as peroxisomal enoyl-CoA isomerase, was found to be present in both peroxisomes and mitochondria. This enzyme seems to be identical with mitochondrial long-chain enoyl-CoA isomerase (Kilponen, J.M., Palosaari, P.M., and Hiltunen, J.K. 1990. Biochem. J. 269, 223-226). All three hepatic enoyl-CoA isomerases have broad chain length specificities but are distinguishable by their preferences for one of the three isomerization reactions. MECI is most active in catalyzing the 3-cis --> 2-trans isomerization; ECI has a preference for the 3-trans --> 2-trans isomerization, and MFE1 is the optimal isomerase for the 2,5 --> 3,5 isomerization. A functional characterization based on substrate specificities and total enoyl-CoA isomerase activities in rat liver leads to the conclusion that the 3-cis --> 2-trans and 2,5 --> 3,5 isomerizations in mitochondria are catalyzed overwhelmingly by MECI, whereas ECI contributes significantly to the 3-trans --> 2-trans isomerization. In peroxisomes, ECI is predicted to be the dominant enzyme for the 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of long-chain intermediates, whereas MFE1 is the key enzyme in the 2,5 --> 3,5 isomerization.     

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.     

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.     

Presence and features of fatty acyl-CoA binding activity in rat hepatic peroxisomes

We studied the fatty acyl-CoA binding activity of rat liver peroxisomes. After subcellular fractionation of rat liver treated with or without clofibrate, a peroxisome proliferator, the binding activity with [1-(14)C]palmitoyl-CoA was detected in the light mitochondrial fraction in addition to the mitochondrial and cytosol fractions. After Nycodenz centrifugation of the light mitochondrial fraction, the binding activity was detected in peroxisomes. The peroxisomal activity depended on the incubation temperature and peroxisome concentration. The activity also depended on the concentration of 2-mercaptoethanol, and a plateau of activity was unexpectedly found at 2-mercaptoethanol concentrations from 20 to 40 mM. Clofibrate increased the total and specific activity of the fatty acyl-CoA binding of peroxisomes by 7.9 and 2.5 times compared with the control, respectively. In the presence of 20% glycerol at 0 degree C, approximately 90% of the binding activity was maintained for up to at least 3 wk. After successive treatment with an ultramembrane Amicon YM series, about 70% of the binding activity was detected in the M.W. 30,000-100,000 fraction. When the M.W. 30,000-100,000 fraction was added to the incubation mixture of the peroxisomal fatty acyl-CoA beta-oxidation system, a slight increase in the beta-oxidation activity was found. 2-Mercaptoethanol (20 mM) significantly activated the fatty acyl-CoA beta-oxidation system to 1.4 times control. After gel filtration of the M.W. 30,000-100,000 fraction, the peaks of fatty acyl-CoA binding protein showed broad elution profiles from 45,000 to 75,000. These results suggest that fatty acyl-CoA binding activity can be detected directly in peroxisomes and is increased by peroxisome proliferators. The high binding activity in the presence of higher concentrations of 2-mercaptoethanol indicates the importance of the SH group for binding. The apparent molecular weight of the binding protein may be from 45,000 to 75,000.     

Existence of acetyl-CoA-dependent chain elongation system in hepatic peroxisomes of rat: effects of clofibrate and di-(2-ethylhexyl)phthalate on the activity

The acetyl-CoA-dependent elongation of medium-chain acyl-CoA in the presence of pyridine nucleotide was studied in rat liver. The activity was increased by the administration of peroxisome proliferators, clofibrate and di-(2-ethylhexyl)phthalate, and the change was more remarkable in peroxisomes than in mitochondria. Addition of 0.01% Triton X-100 to the incubation mixture caused an increase in the mitochondrial activity, whereas the peroxisomal activity did not increase significantly. The pH optimum for the peroxisomal activity was in the range of pH 6.5-7.0 and that for the mitochondrial activity was pH 7.5-8.0. The specificities of primer chain length in both organelles were almost the same, and octanoyl-CoA was the preferred substrate. Peroxisomal activity was completely inhibited by the addition of 1 mM N-ethylmaleimide or 1 mM p-hydroxymercuribenzoic acid, while the activity did not change on the addition of 1 mM KCN or an antibody to acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system. The activity of enoyl-CoA reductase, which catalyzes the last step of the elongation system, was also detected in peroxisomes, although the main activity was localized in microsomes. When the liver peroxisomal fraction of clofibrate-treated rats was incubated with a mixture of octanoyl-CoA, acetyl-CoA, NADH, NADPH, and Triton X-100 in a buffer system, dodecanoyl-CoA was detected as the main product by radio-gas chromatography. On the other hand, the elongation activity was decreased greatly by the addition of NAD+ into the mixture. These results indicate that (i) peroxisomes have activity to elongate medium chain acyl-CoA; (ii) the peroxisomal elongation system may consist of the reverse reaction of the beta-oxidation system except for the last step, which is catalyzed by enoyl-CoA reductase; and (iii) the peroxisomal elongation system is less active than the beta-oxidation system under physiological conditions.     

Crystallization and preliminary X-ray diffraction studies of mitochondrial short-chain delta 3,delta 2-enoyl-CoA isomerase from rat liver.

Crystals of short-chain delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) from rat liver mitochondria have been grown using the hanging-drop vapour diffusion technique. The enoyl-CoA isomerase is an auxiliary enzyme in the beta-oxidation pathway of fatty acid metabolism, and catalyzes the isomerization of unsaturated fatty acids to produce the metabolizable delta 2-trans isomer. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 47.9, b = 118.4 and c = 164.8 A, and diffract to 3 A.     

Intraparticulate localization and some properties of a clofibrate-induced peroxisomal aldehyde dehydrogenase from rat liver

A study was made of the effect of chronic administration of the hypolipidemic drug clofibrate on the activity and intracellular localization of rat liver aldehyde dehydrogenase. The enzyme was assayed using several aliphatic and aromatic aldehydes. Clofibrate treatment caused a 1.5 to 2.3-fold increase in the liver specific aldehyde dehydrogenase activity. The induced enzyme has a high Km for acetaldehyde and was found to be located in peroxisomes and microsomes. Clofibrate did not alter the enzyme activity in the cytoplasmic fraction. The total peroxisomal aldehyde dehydrogenase activity increased 3 to 4-fold under the action of clofibrate. Disruption of the purified peroxisomes by the hypotonic treatment or in the alkaline conditions resulted in the release of catalase from the broken organelles, while aldehyde dehydrogenase as well as nucleoid-bound urate oxidase and the peroxisomal membrane marker NADH:cytochrome c reductase remained in the peroxisomal 'ghosts'. At the same time, treatment by Triton X-100 led to solubilization of the membrane-bound NADH:cytochrome c reductase and aldehyde dehydrogenase from intact peroxisomes and their 'ghosts'. These results indicate that aldehyde dehydrogenase is located in the peroxisomal membrane. The peroxisomal aldehyde dehydrogenase is active with different aliphatic and aromatic aldehydes, except for formaldehyde and glyceraldehyde. The enzyme Km values lie in the millimolar range for acetaldehyde, propionaldehyde, benzaldehyde and phenylacetaldehyde and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the enzyme. Aldehyde dehydrogenase was inhibited by disulfiram, N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic)acid. According to its basic kinetic properties peroxisomal aldehyde dehydrogenase seems to be similar to a clofibrate-induced microsomal enzyme. The functional role of both enzymes in the liver cells is discussed.