Peroxisomal Carnitine Research Articles

Homology modeling and docking studies of Peroxisomal carnitine Ooctanoyltransferase

Homology modeling and docking studies of Peroxisomal carnitine Ooctanoyltransferase

Peroxysomal Carnitine Acetyl Transferase Influences Host Colonization Capacity in Sclerotinia sclerotiorum

In lower eukaryotes, the glyoxylate cycle allows cells to utilize two-carbon compounds when simple sugars are not available. In filamentous fungi, glyoxylate metabolism is coupled with β-oxidation of fatty acids, and both are localized to ubiquitous eukaryotic organelles called peroxisomes. Acetyl coenzyme A (acetyl-CoA) produced during β-oxidation is transported via the cytosol into mitochondria for further metabolism. A peroxisomal-specific pathway for acetyl-CoA transport requiring peroxisomal carnitine acetyl transferase (CAT) activity has been identified in Magnaporthe grisea peroxisomes. Here, we report that a Sclerotinia sclerotiorum ortholog of the M. grisea peroxisomal CAT-encoding gene Pth2 (herein designated Ss-pth2) is required for virulence-associated host colonization. Null (ss-pth2) mutants, obtained by in vitro transposon mutagenesis, failed to utilize fatty acids, acetate, or glycerol as sole carbon sources for growth. Gene expression analysis of these mutants showed altered levels of transcript accumulation for glyoxylate cycle enzymes. Ss-pth2 disruption also affected sclerotial, apothecial, and appressorial development and morphology, as well as oxalic acid accumulation when cultured with acetate or oleic acid as sole carbon nutrient sources. Although mutants were able to penetrate and initially colonize host tissue, subsequent colonization was impaired. Genetic complementation with the wild-type Ss-pth2 restored wild-type virulence phenotypes. These findings suggest an essential role in S. sclerotiorum for the peroxisomal metabolic pathways for oxalic acid synthesis and host colonization.

Modulating aroma compounds during wine fermentation by manipulating carnitine acetyltransferases inSaccharomyces cerevisiae

The wine yeast Saccharomyces cerevisiae is central in the production of aroma compounds during fermentation. Some of the most important yeast-derived aroma compounds produced are esters. The esters ethyl acetate and isoamyl acetate are formed from alcohols and acetyl-CoA in a reaction catalysed by alcohol acetyltransferases. The pool of acetyl-CoA available in yeast cells could play a key role in the development of ester aromas. Carnitine acetyltransferases catalyse the reversible reaction between carnitine and acetyl-CoA to form acetylcarnitine and free CoA. This reaction is important in transferring activated acetyl groups to the mitochondria and in regulating the acetyl-CoA/CoA pools within the cell. We investigated the effect of overexpressing CAT2, which encodes the major mitochondrial and peroxisomal carnitine acetyltransferase, on the formation of esters and other flavour compounds during fermentation. We also overexpressed a modified CAT2 that results in a protein that localizes to the cytosol. In general, the overexpression of both forms of CAT2 resulted in a reduction in ester concentrations, especially in ethyl acetate and isoamyl acetate. We hypothesize that overproduction of Cat2p favours the formation of acetylcarnitine and CoA and therefore limits the precursor for ester production. Carnitine acetyltransferase expression could potentially to be used successfully in order to modulate wine flavour.

Peroxisomal carnitine acetyl transferase is required for elaboration of penetration hyphae during plant infection by Magnaporthe grisea

Magnaporthe grisea, the causal agent of rice blast disease, invades plant tissue due to the action of specialized infection structures called appressoria, which are used to breach the leaf cuticle and allow development of intracellular, infectious hyphae. In this report we demonstrate that peroxisomal carnitine acetyl transferase (CAT) activity is necessary for appressorium function, and in particular, for the elaboration of primary penetration hyphae. The major CAT activity in M. grisea is encoded by the PTH2 gene, which shows elevated expression in response to acetate and lipid, and is regulated by the cyclic AMP response pathway. Furthermore, a Pth2-GFP fusion protein colocalizes with a peroxisomal marker protein. Targeted deletion of PTH2, generated mutants that were completely non-pathogenic, lacked CAT activity and were unable to utilize a range of lipid substrates. The impairment of appressorium function in Deltapth2 was associated with a delay in lipid reserve mobilization from germ tubes into developing infection cells, and abnormal chitin distribution in infection structures. Addition of glucose to Deltapth2 mutants partially restored the ability to cause rice blast disease and lipid reserve mobilization. Taken together, our findings provide evidence that Pth2 plays a role in the generation of acetyl CoA pools necessary for appressorium function and rapid elaboration of penetration hyphae during host infection.

The G553M Mutant of Peroxisomal Carnitine Octanoyltransferase Catalyses Acetyl Transfer and Acetyl-CoA Hydrolysis

The structure of carnitine acetyltransferase revealed a putative binding site for longer acyl chains but access was blocked by methionine 564 (G. Jogl and L. Tong (2003) Cell 112, 113–122). The equivalent residue in all long chain carnitine acyltransferases is a conserved glycine. Mutation of glycine 553 to methionine in bovine COT resulted in loss of activity with all acyl-CoA substrates except acetyl-CoA, supporting the hypothesis that the methionine blocks access for longer acyl chains. The kinetic characteristics of acetyl transfer to carnitine were identical in the native and mutant enzyme. However, rapid acetyl-CoA hydrolysis in the mutant but not the wild-type indicates perturbation of the catalytic site.

Candida albicans CTN gene family is induced during macrophage infection: homology, disruption and phenotypic analysis of CTN3 gene

We have isolated a Candida albicans gene, coding for a putative peroxisomal carnitine acetyl transferase (CTN) protein, which is up-regulated during macrophage infection. In the present study, we describe the disruption of CTN3 gene (previously called CAT3) to gain insight into its potential role during infection. The ability of disrupted Candida mutants to filament was affected by several solid media. Northern blot analysis revealed that CTN3 gene may be involved not only in conditions of cell starvation but also during the process of germination. In agreement with the putative peroxisomal localization of the corresponding protein, we observed a strong glucose repression of CTN3 gene and, on the contrary, high level of transcription by carbon sources that induce the formation of peroxisomal proteins. Furthermore, we showed the existence of two additional C. albicans CTN encoding sequences, which are also induced during macrophage infection.

Structural and mutational characterization of l-carnitine binding to human carnitine acetyltransferase

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Å with an R factor value of 18.9% ( R free=22.3%). l-Carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His 322. The quaternary nitrogen of carnitine forms a π-cation interaction with Phe 545, while Arg 497 forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr 431, Thr 444, and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr 431, Thr 444, Arg 497, and Phe 545 are essential for high affinity binding of l-carnitine.

Inhibition by etomoxir of rat liver carnitine octanoyltransferase is produced through the co-ordinate interaction with two histidine residues

Rat peroxisomal carnitine octanoyltransferase (COT), which facilitates the transport of medium-chain fatty acids through the peroxisomal membrane, is irreversibly inhibited by the hypoglycaemia-inducing drug etomoxir. To identify the molecular basis of this inhibition, cDNAs encoding full-length wild-type COT, two different variant point mutants and one variant double mutant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae, an organism devoid of endogenous COT activity. The recombinant mutated enzymes showed activity towards both carnitine and decanoyl-CoA in the same range as the wild type. Whereas the wild-type version expressed in yeast was inhibited by etomoxir in an identical manner to COT from rat liver peroxisomes, the activity of the enzyme containing the double mutation H131A/H340A was completely insensitive to etomoxir. Individual point mutations H131A and H340A also drastically reduced sensitivity to etomoxir. Taken together, these results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by etomoxir and that the full inhibitory properties of the drug will be shown only if both histidines are intact at the same time. Our data demonstrate that both etomoxir and malonyl-CoA inhibit COT by interacting with the same sites.

Identification of the two histidine residues responsible for the inhibition by malonyl-CoA in peroxisomal carnitine octanoyltransferase from rat liver

Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2–200 μM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.

Sorting of peroxisomal and mitochondrial carnitine acetyltransferase isozymes in the diploid yeast, Candida tropicalis.

Peroxisomal and mitochondrial carnitine acetyltransferase (CAT; EC 2.3.1.7) isozymes are synthesized from the first and second ATG codons of the open reading frame of one gene, Candida tropicalis CAT. Primer extension analysis and RNase protection assay revealed that the peroxisomal CAT, initiating at the second AUG codon of the transcripts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When C. tropicalis CAT was introduced into the other yeast, Saccharomyces cerevisiae, 5' ends of transcripts were similar to those observed in C. tropicalis. Peroxisomal and mitochondrial CAT isozymes were strongly suggested to occur by the alternative initiation of translation, chiefly dependent on the structure or sequence context of the region from the 5' end to the second AUG codon; their transcripts harbored sufficient information to bring about alternative initiation of translation in both yeasts. Sorting of peroxisomal and mitochondrial CAT isozymes to their own compartment was carried out by their own targeting sequences, but, before transportation to their destination, their biosyntheses were regulated by alternative initiation of translation.

Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured fibroblasts

The relationship between peroxisomal and mitochondrial oxidation of the methyl branched fatty acids, phytanic acid and pristanic acid, was studied in normal and mutant human skin fibroblasts with established enzyme deficiencies. Tandem mass spectrometry was used for analysis of the acylcarnitine intermediates. In normal cells, 4,8-dimethylnonanoylcarnitine (C11:0) and 2,6-dimethylheptanoylcarnitine (C9:0) accumulated after incubation with either phytanic acid or pristanic acid. These intermediates were not observed when peroxisome-deficient cells from Zellweger patients were incubated with the same compounds, pointing to the involvement of peroxisomes in the formation of these acylcarnitine intermediates. Similar experiments with fibroblasts deficient in carnitine palmitoyltransferase I, carnitine-acylcarnitine translocase or carnitine palmitoyltransferase II revealed that mitochondrial carnitine palmitoyltransferase I is not required for the oxidation of phytanic acid or pristanic acid, whereas both carnitine-acylcarnitine translocase and carnitine palmitoyltransferase II are necessary. These studies demonstrate that both phytanic acid and pristanic acid are initially oxidized in peroxisomes to 4,8-dimethylnonanoyl-CoA, which is converted to the corresponding acylcarnitine (presumably by peroxisomal carnitine octanoyltransferase), and exported to the mitochondrion. After transport across the mitochondrial membrane and transfer of the acylgroup to coenzyme A, further oxidation to 2,6-dimethylheptanoyl-CoA occurs.—Verhoeven, N. M., D. S. Roe, R. M. Kok, R. J. A. Wanders, C. Jakobs, and C. R. Roe. Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured fibroblasts. J. Lipid Res. 1998. 39: 66–74.

The influence of dietary fatty acids on the activity and metabolic control of peroxisomal carnitine palmitoyltransferase in the liver

Peroxisomes play an important role in the β-oxidation of fatty acids and they possess a membrane-bound carnitine palmitoyltransferase (CPT). Whether this enzyme is regulated by the amount and type of fat consumed in the diet has not been investigated. Therefore weanling male Lewis rats were fed for various times (2 days to 4 weeks) on a low-fat (2.5% w/w) diet (LF) or on high-fat diets containing 20% (w/w) of either hydrogenated coconut oil (HCO), olive oil (OO), safflower oil (SO), evening primrose oil (EPO) or menhaden (fish) oil (MO). Hepatic peroxisomal CPT activity and sensitivity to inhibition by malonyl CoA were examined. In general, feeding the high-fat diets resulted in higher CPT activity per mg peroxisomal protein (specific activity) and per g liver (capacity) than feeding the LF diet throughout the 4 week feeding period. The most rapid induction of CPT specific was seen in the livers of animals fed the SO, EPO and MO diets. Induction of CPT specific activity was maximal after one week of feeding each of the diets. Feeding the MO diet resulted in the highest CPT specific activity at most time points examined and the highest CPT capacity at all time points. In relation to feeding the LF diet, the HCO and EPO diets increased CPT specific activity and capacity by approximately 2-fold while the OO, SO and MO diets increased the CPT specific activity by 3- to 4-fold; these diets increased CPT capacity by 3.5-fold (OO), 4.5-fold (SO) and 7.4-fold (MO). These observations are consistent with previous observations that dietary fish oils increase peroxisomal β-oxidation. Peroxisomal CPT from animals fed the LF diet was the most sensitive to inhibition by malonyl CoA at each time point ([I 50] = approx. 0.5 μM). Each of the high-fat diets decreased the sensitivity of peroxisomal CPT to inhibition by malonyl CoA. The decrease in sensitivity was apparent after feeding the OO, SO or MO diets for 2 days. At all time points peroxisomal CPT from MO-fed rats was the least sensitive to malonyl CoA inhibition. At its least sensitive (after 7 days of feeding) peroxisomal CPT from MO-fed rats required a 5.7-fold higher malonyl CoA concentration to induce 50% inhibition than did the enzyme from LF-fed rats. These data indicate that both the level of fat in the diet and the fatty acid composition of high-fat diets influence CPT activity and its sensitivity to the inhibitor malonyl CoA.

Evidence of two catalytically active carnitine medium/long chain acyltransferases in rat liver peroxisomes

Peroxisomal matrix proteins were extracted from the highly purified peroxisomes with sodium pyrophosphate, and the membranes were sedimented by high speed centrifugation. Biochemical marker enzyme analyses revealed a quantitative release of a number of well-known peroxisomal matrix proteins from the purified peroxisomes. In contrast, carnitine medium/long chain acyltransferase activity, assayed with decanoyl-CoA and palmitoyl-CoA as substrates, was equally distributed in the membrane and the matrix fractions. The matrix and the membrane enzyme activities were differentially affected by a number of detergents. The enzyme in the membrane fraction showed higher malonyl-CoA sensitivity compared to the enzyme in the matrix fraction. The enzyme(s) from the purified peroxisomes or the peroxisomal membranes was quantitatively solubilized by sodium cholate, and the cholate-solubilized enzyme retained malonyl-CoA sensitivity. The membrane enzyme was separated from the matrix enzyme by hydroxylapatite column chromatography. The separation of the membrane enzyme or the matrix enzyme by hydroxylapatite column chromatography resulted in loss of malonyl-CoA sensitivity. The partially purified membrane and the matrix enzymes showed broad substrate specificity, and the highest enzyme activities for both were observed with decanoyl-CoA. In contrast to the matrix enzyme, the membrane enzyme was strongly inhibited by high concentrations (> or = 50 microM) of acyl-CoAs (> 10 carbons in length). The matrix enzyme showed a 2.5-fold lower Km for carnitine compared to the membrane enzyme. The catalytic properties of the partially purified matrix enzyme appear to be similar to the well-characterized peroxisomal carnitine octanoyltransferase, though we find highest activity with decanoyl-CoA rather than octanoyl-CoA as a substrate. The data presented clearly indicate that the membrane and the matrix enzyme activities are due to different proteins.

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.

Molecular cloning and sequence analysis of the rat liver carnitine octanoyltransferase cDNA, its natural gene and the gene promoter

The full-length cDNA and the natural gene for rat peroxisomal carnitine octanoyltransferase (COT) have been isolated and sequenced. The 2681 bp long cDNA contains an open reading frame for 613 amino acids, resulting in a protein with a deduced molecular weight of 70 301, and a C-terminal peroxisomal targeting sequence (Ala-His-Leu). The isolated COT cDNA has 51 bp of the 5′ untranslated region (UTR), 791 bp of 3′ UTR, two putative polyadenylation sites, and a poly(A 19–23) tail. Screening of a rat genomic DNA library in the λ phage with the COT cDNA probe resulted in the isolation of seven overlapping clones, together containing the complete COT gene with seventeen exons. All of the exon-intron boundary sequences conform to the GT-AG rule. The COT gene appears to spread over 40 to 60 kbp region of the rat genome. The transcription initiation site of the COT gene was determined through primer extension, and the promoter sequence up to the position −1140 was established. The promoter lacks the canonical TATA box and a promoter-reporter construct containing the sequence encompassing −1140 to +84 base positions and the firefly luciferase reporter cDNA yielded about 100-fold increase in promoter activity in transfected hepatoma cells. Some of the consensus sequences for putative cis elements present in the promoter sequence are: the two CCAAT motifs for CTF/NF1/CBP binding (at −284 and −93), two GC boxes for Sp1 binding (at −160 and −68), two AP2 sites (at −359 and −25), a half site (TGACCT) for the peroxisome proliferator activated receptor (PPAR) binding at −737 within a partial palindromic sequence region. Potential regulatory elements, such as several palindromes and repeat motifs for five different sequence segments, are also identified.

Peroxisome Proliferation and Microsomal Enzyme Induction by the Hypolipidemic CI-924 in Rats and Mice: Relationship to Tumorgenicity

Peroxisome Proliferation and Microsomal Enzyme Induction by the Hypolipidemic CI-924 in Rats and Mice: Relationship to Tumorgenicity. Hofstra, A. H., King, L. M., and Walker, R. M. (1995). Fundam. Appl. Toxicol. 27, 277-286. CI-924 (5,5′-[[1,1′-biphenyl]-2,5-diylbis(oxy)]bis[2,2-dimethylpentanoic acid]), a lipid lowering agent, was previously shown to be hepatotumorigenic in male and female B6C3F1 mice but not in male and female albino Wistar rats. To determine if the difference between the species in tumorigenic response correlated with the extent of peroxisome proliferation or microsomal changes the effects of CI-924 on liver were characterized in rats and mice. CI-924 doses of 0, 5, 25, 75, and 150 mg/kg were administered in the diet for 4 weeks to B6C3F1 mice and albino Wistar rats. Peroxisomal β-oxidation activity was significantly increased in all groups at doses of 25 mg/kg or higher and was induced up to 25 times in male rats. Peroxisomal carnitine acyltransferase and acyl-CoA oxidase activities were also increased, with the greatest induction observed in male rats. Catalase activity quadrupled in rats and doubled in mice. Serum liver enzyme activities were unchanged with the exception of 5′-nucleotidase which was elevated in mice and decreased in male, but not female, rats. Glutathione S-transferase decreased in the males of both species and glutathione peroxidase increased in the mice. Cytochrome P450 4A1 increased in both species at doses of 25 mg/kg or greater and correlated with increased lauric acid hydroxylation. The high degree of peroxisome proliferation in male rats was unexpected in light of the lack of tumorgenicity demonstrated in a previous 2-year study and these results indicate that early peroxisome proliferation alone is not always a good predictor of hepatocarcinogenicity.

Hepatic microsomal induction profile of carbamic acid [[2,6-bis(1-methylethyl)phenoxy] sulfonyl]-2,6-bis(1-methylethyl) phenyl ester, monosodium salt (PD138142-15), a novel lipid regulating agent

Induction of hepatic microsomal cytochrome P450 produced by carbamic acid [[2,6-bis(1-methylethyl)phenoxy]sulfonyl]-2,6-bis(1-methylethyl) phenyl ester, monosodium salt (PD138142-15), a novel water-soluble inhibitor of acyl-CoA:cholesterol acyltransferase, was examined in male and female rats, dogs, and monkeys, and in male guinea pigs. Relative to control, PD138142-15 increased hepatic microsomal total spectral P450 in all species examined. Hepatic microsomal ethoxyresorufin- O-deethylase, pentoxyresorufin- O-dealkylase, and peroxisomal carnitine acetyltransferase activities and cyanide-insensitive β-oxidation were affected only marginally. Erythromycin- N-demethylase activity was increased (2- to 6-fold) in all three species in which it was examined (rat, dog and pig). Marked increases in immunoreactive P450 3A were noted in rats and dogs, while slight increases were seen in monkeys. Pharmacokinetic studies of PD138142-15 in rats and dogs revealed pronounced decreases (80–90%) in plasma C max, and AUC within 2 weeks of initiation of daily dosing. In spite of the marked decline in plasma drug levels, efficacy in dogs, as determined by serum cholesterol levels, was maintained for up to 6 weeks with continued dosing. Potential acid (gastric) breakdown products of PD138142-15 were examined for their hepatic cytochrome P450 induction profiles in rats and were found to differ both quantitatively and qualitatively from profiles produced by the parent compound. This suggested that induction observed in rats was due to parent PD138142-15 and not to any of the known potential acid breakdown products. The cumulative data establish that PD138142-15 is an inducer of P450 3A in rats and dogs. The results also suggest that P450 3A is induced in monkeys and pigs as well, although the data are less definitive. Decreases in plasma drug levels imply that the compound may be an autoinducer in dogs and rats. The maintenance of efficacy in spite of decreased drug levels in dogs suggests that the effects on serum cholesterol are due to a metabolite or that cholesterol lowering effects occur before the compound is metabolized by the liver.

Regulation of the long-chain carnitine acyltransferases.

Long-chain carnitine acyltransferases are a family of enzymes found in mitochondria, peroxisomes, and endoplasmic reticulum that catalyze the exchange of carnitine for coenzyme A in the fatty acyl-CoA. Conversion of the fatty acyl-CoA to fatty acylcarnitine renders the fatty acid more permeable to the various cellular membranes. The mitochondrial carnitine palmitoyltransferases are considered important in the regulation of mitochondrial beta-oxidation of long-chain fatty acids. However, palmitoylcarnitine produced by peroxisomal carnitine octanoyltransferase or by microsomal carnitine palmitoyltransferase is not different from that produced by the mitochondrial enzyme. Therefore, for there to be control of fatty acid oxidation by the long-chain carnitine acyltransferases, there would have to be some mechanism to coordinately regulate these varied enzymes. The first system of regulation involves inhibition by malonyl-CoA, an intermediate in the synthesis of fatty acids. Malonyl-CoA inhibits long-chain carnitine acyltransferase activity by all three enzymes at similar concentrations in the physiological range. In addition, the mitochondrial and peroxisomal enzymes are known to be regulated at the level of mRNA transcription by a number of shared factors. Although the microsomal enzyme is less well studied, there does, indeed, appear to be a pattern of coordinate regulation for this system.

Effects of Proglycosyn (LY177507) on Fatty Acid Metabolism in Rat Hepatocytes

Proglycosyn (LY177507) belongs to a series of powerful agents that stabilize liver glycogen stores by promoting glycogen synthesis from different precursors and inhibiting glycogenolysis and glycolysis. In the present study we have examined the effects of proglycosyn on fatty acid metabolism in isolated hepatocytes. Preincubation of hepatocytes with medium containing proglycosyn led to a marked inhibition of fatty acid synthesis de novo and acetyl-CoA carboxylase activity without affecting fatty acid synthase. Likewise, proglycosyn depressed the synthesis of triacylglycerols and phospholipids from labeled palmitate. Although octanoate oxidation was unaffected by proglycosyn, mitochondrial palmitate oxidation was notably stimulated. This effect may be attributed to the proglycosyn-induced decrease of intracellular malonyl-CoA levels relative to control incubations and the concomitant relieve of the inhibition of the mitochondrial-outer-membrane carnitine palmitoyltransferase by malonyl-CoA. By contrast, neither peroxisomal palmitate oxidation nor peroxisomal carnitine palmitoyltransferase activity was changed upon hepatocyte incubation with proglycosyn. Results thus indicate that proglycosyn increases the fatty-acid-oxidation efficiency of the liver at the expense of lipogenesis, and this may contribute to the proglycosyn-induced sparing of liver glycogen stores.

Activity of carnitine palmitoyltransferase in mitochondrial outer membranes and peroxisomes in digitonin-permeabilized hepatocytes. Selective modulation of mitochondrial enzyme activity by okadaic acid.

A procedure is described for the rapid measurement of the activity of mitochondrial-outer-membrane carnitine palmitoyltransferase (CPTo) and peroxisomal carnitine palmitoyltransferase (CPTp) in digitonin-permeabilized hepatocytes. CPTo activity was determined as the tetradecylglycidate (TDGA)-sensitive malonyl-CoA-sensitive CPT activity, whereas CPTp activity was monitored as the TDGA-insensitive malonyl-CoA-sensitive CPT activity. Under these experimental conditions, the respective contributions of CPTo and CPTp to total hepatocellular malonyl-CoA-sensitive CPT activity were 74.6 and 25.4%, which correlated well with the values of 76.9 and 23.1% for the respective contributions of the mitochondrial and the peroxisomal compartment to total hepatocellular palmitate oxidation. The sensitivity of CPTo to inhibition by malonyl-CoA was very similar to that of CPTp; thus 50% inhibition of CPTo and CPTp activities was achieved with malonyl-CoA concentrations of 2.6 +/- 0.5 and 3.0 +/- 0.4 microM respectively. Short-term incubation of hepatocytes with the phosphatase inhibitor okadaic acid (i) increased the activity of CPTo and the rate of mitochondrial palmitate oxidation, (ii) decreased the affinity of CPTo for palmitoyl-CoA substrate, and (iii) decreased the sensitivity of CPTo to inhibition by malonyl-CoA. By contrast, neither the properties of CPTp nor the rate of peroxisomal palmitate oxidation were changed upon incubation of cells with okadaic acid. Results indicate therefore that CPTo, but not CPTp, may be regulated by a mechanism of phosphorylation/dephosphorylation. The physiological relevance of these findings is discussed.