The hepatic effects of hypolipidemic drugs (clofibrate, nafenopin, tibric acid, and Wy-14,643) on hepatic peroxisomes and peroxisome-associated enzymes.
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Male Swiss-Webster mice were fed diets containing four hypolipidemic agents which are known to induce proliferation of hepatic peroxisomes. Treatment with all four drugs (clofibrate; its structural analogue, nafenopin; and two drugs structurally unrelated to clofibrate, tibric acid and Wy-14,643) produced a marked hepatomegaly in the mice. The extent of the increase in liver weight correlated well with the increases in total hepatic DNA and in the collective volume of hepatocyte peroxisomes. Treatment with these drugs also produced similar increases in the activities of peroxisome-associated enzymes. The most dramatic increases were noted in the activities of the short-chain (8- to 26-fold) and medium-chain (4- to 11-fold) carnitine acyltransferase. Significant increases were also noted in the activities of catalase (twofold to threefold), alpha-glycerophosphate dehydrogenase (twofold to threefold) and the long-chain carnitine acyltransferase (twofold to fourfold). Activity of the latter enzyme, however, is not known to be associated with peroxisome fractions. Concomitant administration of actinomycin D or cycloheximide with a single oral dose of clofibrate diminished the increases in liver weight and carnitine acyltransferase which occurred with clofibrate treatment alone. The finding that the major increase in activity of peroxisome enzymes occurred in those associated with metabolism of acyl CoA groups supports the hypothesis that the hypolipidemic action of the drugs and the proliferation of hepatic peroxisomes are related functions. (+info)
Identification of a cardiac carnitine binding protein.
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The existence of a cardiac carnitine binding protein was demonstrated using an in vitro binding assay. The binding activity was solubilized with Triton X-100 from the pellet following a 59,000 X g centrifugation of rat ventricular homogenates. Preincubation at temperatures above 40 degrees C or treatment with pronase significantly reduced the binding activity, suggesting that the activity was that of a protein. Cell fractionation studies suggested that the cardiac carnitine binding protein was associated with the plasma membrane fraction and that its activity was distinct from carnitine palmitoyl-transferase, carnitine acetyltransferase, and carnitine translocase. Optimal binding required 60 min of incubation at 25 degrees C. The binding of carnitine to the cardiac carnitine binding protein was saturable, and a dissociation constant of 0.7 microM for DL-carnitine was measured. L-Carnitine competed with DL-[methyl-3H]carnitine for competition by D-carnitine was much less effective. Binding was significantly inhibited when N-ethylmaleimide, iodoacetic acid, or mercuric chloride was present. Once DL-[3H]carnitine was bound to the cardiac carnitine binding protein, radioactivity could be dissociated by a variety of mild treatments including dialysis, overnight incubation at 4 degrees C, and application to a gel filtration column. (+info)
Binding of the enzymes of fatty acid beta-oxidation and some related enzymes to pig heart inner mitochondrial membrane.
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The binding of crotonase (enoyl-CoA hydratase), beta-hydroxyacyl-CoA dehydrogenase, beta-ketothiolase, succinyl-CoA transferase, and carnitine acetyltransferase to inner mitochondrial membranes, mitoplasts, intact mitochondria, erythrocyte membranes, and phosphatidylcholine liposomes was studied. Succinyl-CoA transferase does not bind to any of these membranes. Carnitine acetyltransferase, on the other hand, binds to all of these membranes. The enzymes of fatty acid beta-oxidation, thiolase, crotonase, and beta-hydroxyacyl-CoA dehydrogenase bind to inner membrane, but not to liposomes. The binding shows a moderate dependence on ionic strength (2-200 mM) and pH (6.5-8). These data indicate the possibility of an organization of the enzymes of fatty acid beta-oxidation on the inner mitochondrial membrane, but do not support the idea of an organization of the enzymes of ketone body catabolism. (+info)
Purification and properties of carnitine acetyltransferase from rat liver.
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Carnitine acetyltransferase was purified from rat liver after induction of the enzyme by feeding with di(2-ethylhexyl)phthalate. Two enzyme sources were used: the mitochondrial fraction and the homogenate of the liver. The purification procedure was essentially the same for the two enzyme sources. The enzyme purified from the mitochondrial fraction consisted of two different polypeptides with molecular weights of 36,500 and 27,000, whereas that from the homogenate consisted of one polypeptide with a molecular weight of 67,500. Amino acid compositions and peptide maps of the limited proteolytic products of the two enzyme preparations were nearly the same. Their antibodies were cross-reactive. Catalytic properties of the two preparations were nearly the same: the specific enzyme activities, double reciprocal plots of initial velocity study, substrate specificities for acylcarnitines having various carbon chain lengths, apparent Michaelis constants for substrates. On electrophoresis of the immunoprecipitate obtained after incubation of the mitochondrial extract, the two immunoreactive polypeptides with molecular weights of 36,500 and 27,000 were found. But only one polypeptide, with molecular weight of 67,500, was detected when the protease inhibitors were added to the mitochondrial extract. It was concluded that the enzyme in the mitochondrial fraction was a monomeric form but was converted into a dimeric form by proteolytic modification after the disruption of mitochondria. The preparation from the post-mitochondrial fraction, which had a lower specific activity, contained two polypeptides whose molecular weights were 69,000 and 67,500. They could not be separated from each other throughout the purification. The peptide maps of the products of the limited proteolysis were very similar. (+info)
Properties of purified carnitine acyltransferases of mouse liver peroxisomes.
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The purpose of this study was to characterize the physical, kinetic, and immunological properties of carnitine acyltransferases purified from mouse liver peroxisomes. Peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase were purified to apparent homogeneity from livers of mice fed a diet containing the hypolipidemic drug Wy-14,643 [( 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]-acetic acid). Both enzymes have a molecular weight of 60,000 and a similar pH optimum. Carnitine octanoyltransferase had a maximum activity for C6 moieties while the maximum for carnitine acetyltransferase was with C3 and C4 moieties. The apparent Km values were between 2 and 20 microM for the preferred acyl-CoA substrates, and the Km values for L-carnitine varied depending on the acyl-CoA cosubstrates used. The Hill coefficient, n, was approximately 1 for all acyl-CoAs tested, indicating Michaelis-Menten kinetics. Carnitine octanoyltransferase retained its maximum activity when preincubated with 5,5'-dithiobis-(2-nitrobenzoate) at pH 7.0 or 8.5. Neither carnitine octanoyltransferase nor carnitine acetyltransferase were inhibited by malonyl-CoA. The immunology of carnitine octanoyltransferase is discussed. These data indicate that peroxisomal carnitine octanoyltransferase and carnitine acetyltransferase function in vivo in the direction of acylcarnitine formation, and suggest that the concentration of L-carnitine could influence the specificity for different acyl-CoA substrates. (+info)
Characterization of peroxisomal and mitochondrial carnitine acetyltransferases purified from alkane-grown Candida tropicalis.
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Properties of peroxisomal and mitochondrial carnitine acetyltransferases purified from an alkane-grown yeast, Candida tropicalis, were compared each other. The molecular weight of both enzymes was estimated to be about 420 000 by analytical ultracentrifugation and gel filtration chromatography with Sepharose 6B. However, each enzyme gave two subunits on the polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate: the peroxisomal enzyme (64 000 and 57 000) and the mitochondrial enzyme (64 000 and 52 000). The subcellularly distinct enzymes gave a similar amino acid composition except for the contents of some amino acids: glycine, valine, glutamic acid and aspartic acid. Their isoelectric point was somewhat different: 5.11 for the peroxisomal enzyme and 5.22 for the mitochondrial enzyme. Both enzymes had the same amino-terminal residue (glutamic acid or glutamine) and the heat stability, and was indistinguishable immunochemically. These results suggest that peroxisomal and mitochondrial carnitine acetyltransferases of C. tropicalis cells may be products of the same nuclear gene. Differences in the molecular weight of the subunits of the enzymes would result from modification or processing of the common protein in the step of distribution to the respective organelles, that is, so-called post-translational modification. (+info)
Synthesis in vitro of precursor-type carnitine acetyltransferase with messenger RNA from Candida tropicalis.
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Carnitine acetyltransferase was synthesized in vitro in the mRNA-dependent reticulocyte system with mRNA from alkane-grown or propionate-grown cells of Candida tropicalis. The protein synthesized in vitro was isolated by immunoprecipitation with antibody against peroxisomal or mitochondrial carnitine acetyltransferase and was compared with peroxisomal carnitine acetyltransferase (Mr of subunits, 64 000 and 57 000) and the mitochondrial enzyme (Mr of subunits, 64 000 and 52 000) of C. tropicalis by electrophoresis in the presence of sodium dodecyl sulfate. Nascent carnitine acetyltransferase prepared in vitro showed a hetero-oligomeric property, like the peroxisomal and mitochondrial enzymes isolated from C. tropicalis. The molecular weights of the subunits of nascent carnitine acetyltransferase were estimated to be 71 000 and 57 000, indicating the existence of the precursor form of the enzyme. By sucrose density gradient centrifugation of total mRNA, these two subunit proteins were shown to be synthesized with respective mRNAs of different sizes. The same precursor-type of carnitine acetyltransferase was obtained with the mRNAs from the alkane-grown cells and the propionate-grown cells. The results obtained suggest that a common precursor will be post-translationally modified to form the peroxisomal and mitochondrial enzymes. (+info)
Induction of carnitine acetyltransferase by clofibrate in rat liver.
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Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine O-acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold. (+info)