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Background: . Plant nonspecific lipid-transfer proteins (nsLTPs) bind a variety of very different lipids in vitro, including phospholipids, glycolipids, fatty acids and acyl coenzyme As. In this study we have determined the structure of a nsLTP complexed with palmitoyl coenzyme A (PCoA) in order to further our understanding of the structural mechanism of the broad specificity of these proteins and its relation to the function of nsLTPs in vivo., Results: . 1H and 13C nuclear magnetic resonance spectroscopy (NMR) have been used to study the complex between a nsLTP isolated from barley seeds (bLTP) and the ligand PCoA. The resonances of 97% of the 1H atoms were assigned for the complexed bLTP and nearly all of the resonances were assigned in the bound PCoA ligand. The palmitoyl chain of the ligand was uniformly 13C-labelled allowing the two ends of the hydrocarbon chain to be assigned. The comparison of a subset of 20 calculated structures to an average structure showed root mean square deviations of 1.89 +/- 0.19 for all C, N, O, P and S atoms of the entire complex and of 0.57 +/- 0.09 for the peptide backbone atoms of the four alpha helices of the complexed bLTP. The four-helix topology of the uncomplexed bLTP is maintained in the complexed form of the protein. The bLTP only binds the hydrophobic parts of PCoA with the rest of the ligand remaining exposed to the solvent. The palmitoyl chain moiety of the ligand is placed in the interior of the protein and bent in a U-shape. This part of the ligand is completely buried within a hydrophobic pocket of the protein., Conclusions: . A comparison of the structures of bLTP in the free and bound forms suggests that bLTP can accommodate long olefinic ligands by expansion of the hydrophobic binding site. This expansion is achieved by a bend of one helix, HA, and by conformational changes in both the C terminus and helix HC. This mode of binding is different from that seen in the structure of maize nsLTP in complex with palmitic acid, where binding of the ligand is not associated with structural changes.

The stromal glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15) from spinach (Spinacia oleracea) and squash (Cucurbita moschata) were expressed in Escherichia coli and their activities with palmitoyl-CoA and oleoyl-CoA compared. The GPAT from squash, a chilling-sensitive plant, was found to have the greatest difference in activities between the two substrates, using palmitoyl-CoA over three times faster than oleoyl-CoA. In contrast, the enzyme from spinach, a chilling-tolerant plant, preferred oleoyl-CoA over palmitoyl-CoA. By using conserved restriction endonuclease sites each of the two genes was divided into three fragments of roughly equal size and recombined to create six different chimeras. All chimeras retained a large portion of their original activity but in most cases the specificity was greatly altered. The central third of the protein was found to contain the structural features which determine substrate specificity of the wild-type GPATs. Two of the chimeras, which have a spinach-derived central region and a squash-derived carboxyl region, were found to have greatly enhanced specificities for 18:1 acyl chains, potentially making them ideal for decreasing the level of saturation of plant membrane lipids through genetic engineering.

Male and female CD rats were administered one of two dose levels of clofibrate, gemfibrozil, or bezafibrate daily by oral gavage for a period of 14 days in order to establish an empirical data base using the Charles River CD rat with a single class of drugs against which the potency of novel proprietary compounds could be compared. Subsequent gross examination of the liver indicated significant and dose-related increases in relative and absolute liver weights in males following clofibrate and gemfibrozil. In females, absolute and relative liver weights were significantly elevated to a similar degree with either dose of gemfibrozil, and absolute liver weights were higher in clofibrate-dosed animals. Bezafibrate had no effect on female liver weights. Clofibrate and gemfibrozil increased hepatic palmitoyl CoA beta-oxidation in both sexes; however, clofibrate had the greater effect in males and gemfibrozil had the least effect in females. Bezafibrate treatment resulted in a very pronounced elevation of palmitoyl CoA beta-oxidation in the males but had no similar effect in the females. Concurrent ELISA analysis for cytochrome CYP4A revealed very good correspondence between beta-oxidation and cytochrome induction for each of the three compounds in males, but other cytochromes were not greatly affected, except CYP1A1 which was elevated in bezafibrate-dosed females. For males, further analysis for markers of cellular proliferation, namely cyclin-dependent kinases (CDK) and proliferating cell nuclear antigen (PCNA), indicated dose-related increases for both with clofibrate, increases at the high dose for gemfibrozil, and, for PCNA, a dose-related increase for bezafibrate. In females, both markers for cell proliferation showed either slight or no increases following any of the three drug treatments. These results demonstrate clear sex-dependent differences in terms of relative potency in the hepatic response of the Sprague-Dawley-derived rat to these peroxisome proliferators. Bezafibrate is most potent and gemfibrozil is least potent in stimulating peroxisome-associated beta-oxidation and cytochrome P450 4A induction in the males. Even though gemfibrozil significantly increased liver weights, beta-oxidation and cytochrome P450 4A in the females increased only after clofibrate treatment, although to a lesser degree than in the males administered the same dose. Similar sex-related differences were observed for cell proliferation. In conclusion, sex-related differences were noted in the potency to stimulate acyl Co-A oxidation, its association with hepatomegaly, and the stimulation of cell proliferation, but CYP4A induction always accompanied any substantial drug-dependent increases in beta-oxidation.

In vivo rates of incorporation and turnover of palmitate and arachidonate in brain phospholipids were measured in awake rats treated chronically with lithium, following intravenous infusion of radiolabeled palmitate and arachidonate, respectively. Chronic lithium, at a brain level considered to be therapeutic in humans, decreased turnover of arachidonate within brain phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine by up to 80% (P < 0.001). In contrast, lithium had a minimal effect on turnover of palmitate, causing only a 26% reduction in turnover in phosphatidylcholine (P < 0.01). These results suggest that a major therapeutic effect of lithium is to reduce turnover specifically of arachidonate, possibly by inhibiting phospholipase A2 involved in signal transduction. The effect may be secondary to the known action of lithium on the phosphoinositide cycle, by inhibiting the activity of inositol monophosphatase.

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.

After incubating isolated rat hepatocytes with [1-14 C]palmitic acid, CoA and ATP (+MgCl2), a significant amount of [1-14 C]palmitoyl-CoA was found in the incubation medium. There was no correlation between its rate of synthesis and the degree of intactness of the cells. The results indicate that there is a long-chain fatty acyl-CoA synthetase active on the external surface of the hepatocyte plasma membrane. The activity of this enzyme was negligible in primary cultures of rat hepatocytes, suggesting that the exofacial long-chain acyl-CoA synthetase is an artifact of the collagenase perfusion technique used to prepare the hepatocytes.

The activity of hepatic fatty acid oxidation enzymes in rats fed linseed and perilla oils rich in alpha-linolenic acid (alpha-18:3) was compared to that in rats fed safflower oil rich in linoleic acid (18:2) and a saturated fat (palm oil). Palm and safflower oils were essentially devoid of alpha-18:3. The palmitoyl-CoA oxidation rates both in mitochondrial and peroxisomal pathways in liver homogenates were significantly higher in rats fed linseed oil than in those fed palm and safflower oils. Among rats fed diets containing palm oil, safflower oil, fat mixtures composed of safflower and perilla oils (2:1, w/w and 1:2, w/w), and perilla oil, mitochondrial and peroxisomal fatty oxidation rates increased with increasing dietary levels of perilla oil. Compared to palm and safflower oils, dietary alpha-18:3 either in the form of linseed or perilla oils profoundly increased the activity of carnitine palmitoyltransferase, acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase. Smaller but significant increases by dietary alpha-18:3 of the activity of acyl-CoA dehydrogenase, enoyl-CoA hydratase, and delta 3, delta 2-enoyl-CoA isomerase were also observed. Unexpectedly, dietary alpha-18:3 greatly reduced the activity of 3-hydroxy-acyl-CoA dehydrogenase. Compared to palm oil, dietary polyunsaturated fats significantly reduced the activity of fatty acid synthetase and glucose-6-phosphate dehydrogenase to the same levels. The activity of pyruvate kinase was significantly higher in rats fed palm oil than in those fed polyunsaturated fats. The extent of reduction was more prominent with polyunsaturated fats containing alpha-18:3 than with safflower oil devoid of alpha-18:3. Thus, compared to linoleic acid and saturated fatty acids, dietary alpha-18:3 caused characteristic changes in the activity of hepatic enzymes in fatty acid and glucose metabolism in rats.

Two critical steps in the assembly of yeast and mammalian glycosylphosphatidylinositol (GPI) anchor precursors are palmitoylation of the inositol residue and mannosylation of the glucosamine residue of the glucosaminyl phosphatidylinositol (GlcNalpha-PI) intermediate. Palmitoylation has been reported to be acyl-CoA dependent in yeast membranes (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 267, 8599-8603) but strictly acyl-CoA independent in rodent membranes (Stevens, V. L., and Zhang, H. (1994) J. Biol. Chem. 269, 31397-31403), and thus poorly conserved. In addition, it was suggested that acylation must precede mannosylation in both yeast (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 276, 8599-8603) and rodent (Urakaze, M., Kamitani, T., DeGasperi, R., Sugiyama, E., Chang, H.-M., Warren, C. D., and Yeh, E. T. H. (1992) J. Biol. Chem. 267, 6459-6462) cells because GlcNalpha-acyl-PI accumulates in vivo when mannosylation is blocked. However, GlcNalpha-acyl-PI accumulation would also be expected if mannosylation and acylation were independent of each other. These issues were addressed by the use of a synthetic dioctanoyl GlcNalpha-PI analogue (GlcNalpha-PI(C8)) as an in vitro substrate for GPI-synthesizing enzymes in Chinese hamster ovary cell membranes. GlcNalpha-PI(C8) was acylated in an manner requiring acyl-CoA. Thus, the process involving acyl-CoA reported for yeast has been conserved in mammals. Furthermore, both GlcNalpha-PI(C8) and GlcNalpha-acyl-PI(C8) could be mannosylated in vitro, but mannosylation of the latter was significantly more efficient. This provides direct support for the earlier suggestion that acylation precedes mannosylation in rodents cells. A similar result was also observed with the Saccharomyces cerevisiae mannosyltransferase. In contrast, it has been reported that mannosylation of endogenous GlcNalpha-PI by Trypansoma brucei membranes occurs without prior acylation. The same result was obtained with GlcNalpha-PI(C8), confirming that the mannosyltransferase of trypanosomes is divergent from those in yeasts and rodents.

We examined the potential of overt carnitine palmitoyltransferase (CPT I) to control the hepatic catabolism of palmitoyl-CoA in suckling and adult rats, using a conceptually simplified model of fatty acid oxidation and ketogenesis. By applying top-down control analysis, we quantified the control exerted by CPT I over total carbon flux from palmitoyl-CoA to ketone bodies and carbon dioxide. Our results show that in both suckling and adult rat, CPT I exerts very significant control over the pathways under investigation. However, under the sets of conditions we studied, less control is exerted by CPT I over total carbon flux in mitochondria isolated from suckling rats than in those isolated from adult rats. Furthermore the flux control coefficient of CPT I changes with malonyl-CoA concentration and ATP turnover rate.

Mammalian H-Ras and N-Ras are GTP-binding proteins that must be post-translationally lipidated to function as molecular switches in signal transduction cascades controlling cell growth and differentiation. These proteins contain a C-terminal farnesyl-cysteine alpha-methyl ester and palmitoyl groups attached to nearby cysteines. Data is presented showing that rat liver microsomes contain an enzyme that transfers the palmitoyl group from palmitoyl-coenzyme A to cysteine residues of H-Ras protein and of a synthetic peptide having the structure of the C terminus of N-Ras. This protein palmitoyltransferase (PPT) was solubilized from membranes and purified 10,500-fold to apparent homogeneity with an overall yield of 10%. On an SDS gel, PPT appears as two proteins of molecular masses of approximately 30 and approximately 33 kDa. If the palmitoylation sites of the N-Ras peptide (the non-farnesylated cysteine) or H-Ras protein (cysteines 181 and 184) are changed to serine, palmitoylation by PPT does not occur. Non-farnesylated H-Ras produced in bacteria as well as in vitro farnesylated bacterial H-Ras are not substrates for PPT nor is the non-farnesylated, methylated N-Ras peptide. These results suggest, but do not prove, that farnesylation and possibly C-terminal methylation are prerequisites for Ras palmitoylation. PPT shows a large preference for palmitoyl-coenzyme A over myristoyl-coenzyme as the acyl donor. Values of Km for palmitoyl-CoA and H-Ras are 4.3 +/- 1.2 and 0.8 +/- 0.3 microM, respectively. PPT is the first protein palmitoyltransferase to be purified, and the availability of pure enzyme should contribute to our understanding of the function and regulation of Ras palmitoylation in cells.

The palmitoylation or S-acylation of at least some G protein alpha subunits is a dynamic process that is regulated in vivo by the activation of associated receptors. Highly purified, myristoylated Gialpha1 and other G protein alpha subunits react spontaneously with palmitoyl-CoA in vitro to form thioesterified proteins. This reaction requires native Gialpha1 and occurs exclusively at Cys3, the same residue that is palmitoylated in vivo. The reaction proceeds to completion, and its rate is roughly equal to the rate of loss of palmitate observed in pulse-chase experiments in vivo. The rate of autoacylation is significantly enhanced by the G protein betagamma subunit complex. Autoacylation may play a role in the dynamic thioesterification of some cellular proteins.

The kinetic properties of the purified trifunctional beta-oxidation complex (TOC) from pig heart mitochondria were analyzed with the aim of elucidating the functional consequence of having three sequentially acting enzymes of beta-oxidation associated in one complex. The kinetic parameters of TOC and of the component enzymes of TOC, long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase, were determined with substrates having acyl chains with 16 carbon atoms. Quantification by high performance liquid chromatography of intermediates formed during the degradation of 2-trans-hexadecanoyl-CoA to myristoyl-CoA and acetyl-CoA by TOC revealed the accumulation of 3-hydroxyhexadecanoyl-CoA, whereas 3-ketohexadecanoyl-CoA was undetectable. The observed rates of NADH and acetyl-CoA formation were higher than the theoretical rates calculated by use of the kinetic parameters and measured concentrations of intermediates. When the sequence of reactions catalyzed by TOC was inhibited by acetyl-CoA, the steady-state concentration of the 3-hydroxyacyl-CoA intermediate was not affected, whereas a small amount of 3-ketohexadecanoyl-CoA was detected. The differences between observed and predicted reaction rates and between measured and expected concentrations of intermediates are best explained by the operation of a channeling mechanism. As a consequence of intermediate channeling between the active sites on the complex, more coenzyme A is available in the mitochondrial matrix and metabolites like 3-ketoacyl-CoA thioesters, which are strong inhibitors of several beta-oxidation enzymes, do not accumulate.

In order to gain a better understanding of the kinetics of activation and inhibition of hepatic monoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22) by fatty acid, we examined the effect of fatty acid with respect to MGAT's long-chain acyl-CoA substrate in Triton X-100 mixed micelles. At concentrations between 2.5 and 5.3 mol %, oleic acid stimulated MGAT activity 2-fold, whereas oleic acid inhibited MGAT at concentrations higher than 7.5 mol %. The dependence on palmitoyl-CoA was highly cooperative with a Hill constant of greater than 2.4. When present at less than 3 mol%, oleic acid eliminated the lag in the dependence curve. When concentrations of oleic acid were higher than 3 mol %, Michaelis-Menton kinetics were observed with an apparent k(m) value of about 54 microM for palmitoyl-CoA but with progressively decreasing Vmax values. This effect was not observed with octanoic acid, suggesting that the medium-chain fatty acid is unable to associate stably with the mixed micelle and, thus, cannot substantially alter substrate affinity. When anionic phospholipids were tested, phosphatidic acid, lysophosphatidic acid, phosphatidylserine, and phosphatidylinositol eliminated some of the lag in activation by palmitoyl-CoA. At high molar concentrations of the anionic lipid activators, apparent k(m) values ranged from 77 microM for phosphatidic acid to 196 microM for phosphatidylinositol. Zwitterionic phospholipids had no effect, nor did the non-phospholipid activators bovine serum albumin or sn-1,2-diacylglycerol. CaCl2, but not neomycin or KC1, could overcome the inhibitory effect of oleic acid; thus, the inhibitory effect of fatty acid did not appear to occur by electrostatic interactions. These blockers did not change the effects observed with the anionic phospholipid activators or with the inhibitor, sphingosine. An altered k(m) for palmitoyl-CoA in the presence of fatty acid or anionic phospholipid suggests that both long-chain fatty acids and phospholipid cofactors may induce a conformational change in MGAT, thereby altering the enzyme's affinity for its long-chain acyl-CoA substrate. These data further support the hypothesis that the synthesis of glycerolipids via the monoacylglycerol pathway may be highly regulated via a variety of lipid second messengers such as phosphatidic acid and diacylglycerol, as well as by the influx of fatty acids derived from high-fat diets, or from the hydrolysis of adipocyte triacylglycerol during fasting or diabetes.

In the silkworm, Bombyx mori, production of the sex pheromone bombykol is regulated by a neurohormone termed PBAN. We have detected the activity of acyl CoA reductase in the pheromone gland of B. mori by using palmitoyl CoA as a substrate. The acyl CoA reductase requires NADPH, but not NADH, as a proton dono. When the pheromone gland was incubated with the PBAN fragment peptide TKYFSPRLamide, palmitoyl CoA was incorporated and converted into the corresponding C16 alcohols. Radio HPLC analysis revealed that these C16 alcohols were hexadecan-1-ol (81.2%), (Z)-11-hexadecen-1-ol (12.3%), and (E, Z)-10, 12-hexadecadien-1-ol (= bombykol, 6.5%). The production of C16 alcohols in the pheromone gland was inhibited by the known bombykol biosynthesis inhibitors EDTA, LaCl3, W-7, trifluoperazine, p-nitrophenyl phosphate, NaF and compactin. By contrast, when the pheromone gland homogenate was incubated in the presence of palmitoyl CoA and NADPH, production of C16 alcohols was affected by compactin, W-7 and trifluoperazine, but not by EDTA, LaCl3, p-nitrophenyl phosphate and NaF. These results indicate that compactin, W-7 and trifluoperazine directly suppress the step catalyzed by acyl CoA reductase, whereas EDTA, LaCl3, pNPP, and NaF inhibit bombykol production by affecting other biochemical steps in the signal transduction of PBAN action. The present results also imply that PBAN regulates the step catalyzed by acyl CoA reductase and that palmitoyl CoA could be used as a substrate of the acyl CoA reductase that regulates bombykol biosynthesis.

Rats were treated with various peroxisome proliferators and concomitant changes in ubiquinone levels were monitored. In addition to clofibrate and di(2-ethylhexyl)phthalate, acetylsalicylic acid, 2-ethylhexanoic acid, thyroxine and dehydroepiandrosterone were used as proliferators. Administration of these compounds increased the contents of ubiquinone in liver and, to some extent, in kidney and muscle. No change in corresponding valued for heart or brain were observed. The treatments did not influence cholesterol levels, but increased the amounts of dolichol in the liver to various extents. Treatment of rats with the catalase inhibitor aminotriazole increased the ubiquinone levels in kidney, heart and muscle but not in liver. Comparison of peroxisomal fatty acid beta-oxidation with ubiquinone amounts in liver homogenates after treatment with a number of peroxisome proliferators demonstrated a direct correlation between these two parameters. Subcellular fractionation of liver after peroxisome proliferation revealed that the ubiquinone level was increased in mitochondria and lysosomes which are the main compartments for this lipid, but an increase was also observed in both peroxisomes and microsomes. The increase in hepatic ubiquinone after treatment with various types of proliferators was related to the decrease in blood cholesterol level. These results show that the volume of the peroxisomal compartment and the ubiquinone content in animal tissues are interrelated.

Data on the subchronic toxicity of 2-ethylhexanol (2EH) were required to establish the dose vehicle and dose levels for oncogenicity studies. In preliminary studies 2EH was given subacutely (11 days) to male and female Fischer 344 rats and B6C3F1 mice as an aqueous emulsion by oral gavage (0, 100, 330, 1000, and 1500 mg/kg/day). Clinical observations were made, body weights, food consumption, clinical chemistries, hematologies, and selected organ weights were measured, and gross and micropathologies were performed. Target organs were the central nervous system, liver, forestomach, spleen, thymus, and kidney in rats and the central nervous system, liver, and forestomach in mice. 2EH was then administered by oral gavage to male and female F344 rats and B6C3F1 mice as an aqueous emulsion (0, 25, 125, 250, and 500 mg/kg/day) for 13 weeks. At 500 mg/kg/day in the rat there was reduced body weight gain (6% male, 7% female), increased relative liver (29% male, 15% female), kidney (16% male, 6% female), stomach (11% male, 16% female), and testes (6%) weights, and moderate gross and microscopic changes in the liver and forestomach. There were no behavioral effects or effects on the spleen or thymus. A no-effect level for target organ effects in the rat was 125 mg of 2EH/kg/day. At 500 mg of 2EH/kg/day in the mouse the only effects were increased relative stomach weights in males (13%) and a low incidence of gross and microscopic findings in the forestomach (male and female) and liver (female). A no-effect level for target organ effects in the mouse was 125 mg of 2EH/kg/day. 2EH was a peroxisome proliferator in the rat but not in the mouse at subchronic dose levels of 500 mg/kg/day. Dose levels in oncogenicity studies were set at 50 mg/kg/day for the absence of treatment-related effects in rats and mice, and 500 and 750 mg/kg/day, respectively, in rats and mice as high doses producing minimal toxicity without altering the life span.

In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-3H]palmitate to maintain constant plasma specific activity. Animals were killed after 2-10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-3H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with > 50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6-1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by approximately 60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.

Vitamin A-deficient male mice were treated for 10 days with 0.02% perfluorooctanoic acid (PFOA) in their diet. The effects of this highly potent peroxisome proliferator on peroxisomal palmitoyl-CoA oxidation and lauroyl-CoA oxidase activities were reduced by 3.1-7.5 times in comparison to the values obtained with mice receiving a vitamin A-adequate diet. The activity of peroxisomal catalase was virtually unaffected by vitamin A deficiency and treatment with PFOA had no significant effect on this activity in vitamin A-adequate or vitamin A-deficient mice. However, vitamin A deficiency itself caused a more than 800% increase in cytosolic catalase activity. Thus, the percentage increase caused by PFOA on cytosolic catalase activity was reduced 12.6 times in vitamin A-deficient mice compared to the increase in vitamin A-adequate mice, although the total absolute activities were similar. These findings suggest that the peroxisome proliferation caused by this peroxisome proliferator is highly dependent on the vitamin A status of the mouse.

P0, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys153. To gain insights into the mechanism of P0 acylation, the in vitro palmitoylation of both P0 and a synthetic Cys153-containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P0 with [3H]palmitoyl-CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P0 was not affected by boiling the isolated P0 for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonenzymatic nature of the reaction. After chemical deacylation, P0 was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [14C]palmitoyl-CoA, the synthetic peptide encompassing the natural P0 acylation site (I150RYCWLRR157) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37 degrees C, peptide palmitoylation followed a second-order reaction (k2 = 246 +/- 6 M-1 min-1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl-CoA. The activation energy calculated from the Arrhenius plot is approximately 2 kcal/mol and much lower than that of enzyme-catalyzed transacylations. Finally, two other P0 peptides (V121PTRYG126 and K109TSQVTL115) as well as various unrelated thiol-containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.

Acyl-coenzyme A (CoA) binding protein (ACBP) is a 10-kd protein that binds acyl-CoA moieties and stimulates medium-chain fatty acid synthesis by goat mammary gland fatty acid synthetase. Its exact role in intermediary lipid metabolism has not been fully elucidated. It is hypothesized that ACBP is directly involved in the metabolism of lipid. In the present study, purified rat liver ACBP was used to generate a polyclonal antisera for radioimmunoassay of ACBP in tissue specimens isolated from fasted rats and rats fed normal rat chow and a high-fat diet. In addition, purified ACBP was used to examine its effect on the activity of mitochondrial outer membrane (OM) carnitine palmitoyltransferase (CPT0). Fasting for 24 hours significantly decreased tissue levels of ACBP in the liver (69.0 +/- 7.2 v 46.7 +/- 5.0 pg/ng DNA), whereas feeding of a high-fat diet for 48 hours caused ACBP levels to increase (69.0 +/- 7.2 v 103.9 +/- 18.0). Hepatic levels of this protein continued to increase and remained elevated with prolonged exposure to the high-fat diet (28 days). A similar pattern of change was observed in the kidney, but the magnitude of change was less. Heart ACBP did not respond acutely to the high-fat diet, but did increase after prolonged exposure (28 days). Fasting had no effect on ACBP levels in kidney and heart. Addition of ACBP to an in vitro assay system significantly increased the activity of CPT0 (from 5.2 +/- 0.8 to 72.1 +/- 5.3 nmol palmitoylcarnitine formed.min-1.mg-1 protein) when measured under inhibiting concentrations of palmitoyl-CoA (40 mumol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty acyl-coenzyme A's are temporarily converted into fatty acylcarnitines while transferred across the inner mitochondrial membrane, in their catabolic pathway. In search of an explanation for the need of this coenzyme exchange, the present work describes comparatively the abilities of both kinds of fatty acyl derivatives (represented by palmitoyl-coenzyme A and palmitoylcarnitine) in binding to and perturbing the structure of phosphatidylcholine bilayers in the form of large unilamellar vesicles. Both palmitoyl-coenzyme A and palmitoylcarnitine partition preferentially into the bilayer lipids, so that their free concentration in water is in practice negligible. However, palmitoylcarnitine is able to disrupt the membrane barrier to solutes, leading to vesicle leakage, and, at higher concentrations, it produces complete membrane solubilization, while palmitoyl-coenzyme A produces neither leakage nor solubilization. Palmitoylcarnitine has the properties of many commonly used biochemical detergents. The different behavior of both fatty acyl derivatives helps to explain the need for the transitory coenzyme A/carnitine exchange, and provides a pathogenic mechanism for some genetic defects of mitochondrial fatty acid transport. Other pathophysiological processes in which palmitoylcarnitine has been putatively involved are examined in light of the above results.

Human erythrocyte ghosts were shown to have palmitoylating activity which acylates both endogenous ghost polypeptides and exogenous proteins derived from Semliki Forest virus (SFV). Cell-free fatty acid transfer from [3H]palmitoyl-CoA to endogenous protein was greatly enhanced in ghosts when pre-existing fatty acids linked to the endogenous acyl proteins were removed by hydroxylamine treatment prior to the transfer reaction. In contrast to erythrocyte acyl proteins acceptor proteins present in human placental membranes were palmitoylated in vitro to a similar extent with or without prior deacylation by hydroxylamine treatment. This indicates the presence of large pools of non-acylated proteins in placenta and small pools in erythrocytes. In testing for the protein substrate specificity of the palmitoyl transferase (PAT) present in ghosts we found that the SFV acceptor proteins, which are totally unrelated to erythrocytes, competed with the palmitoylation of endogenous ghost protein acceptors. This palmitoylating enzyme is inhibited by Cibacron Blue, SDS, and heat treatment, but stimulated in the presence of low concentrations of mild detergent (TX-100). Since PAT operating at the surface membrane of red blood cells has properties very similar to those of PAT present in human placental microsomes [1], we suggest that only one type of PAT may transfer fatty acids to various acylproteins that occur at multiple locations in different tissues [2].

The finding that the intracellular storage material in juvenile neuronal ceroid lipofuscinosis (JNCL) consists of the subunit c of ATP synthase prompted us to study energy conservation in JNCL patients. The activities of respiratory chain enzymes in isolated muscle mitochondria from 8 JNCL cases were normal, but oxidation of palmitate was reduced in 6 patients. The degree of reduction was related to the age of the patients. None of the patients had clinical symptoms or laboratory findings of impaired energy conservation, which suggest that the reduced palmitate oxidation was not associated with a major defect in fatty acid oxidation.

Negative chemical ionization (NCI) mass spectrometry was used to quantify the acyl-CoA intermediates present in human fibroblasts growing in media containing the long-chain fatty acid, palmitate. The acyl-CoA intermediates were detected as the N-acyl pentafluorobenzyl glycinates. In fibroblasts from normal individuals only saturated acyl-CoA esters were detected, supporting the concept that the acyl-CoA dehydrogenase reaction is the rate-limiting step of intramitochondrial fatty acid oxidation. In patients with inherited enzymatic defects of intramitochondrial long-chain fatty acid oxidation, there was not a significant increase in the amount of long-chain acyl-CoA compounds, with palmitoyl-CoA amounts similar to those found in controls. However, there was a sharp decrease in the relative amount of lauroyl-CoA and a resultant sixfold elevation in the palmitoyl-CoA:lauroyl-CoA ratio. In contrast, fibroblasts with a defect involving the transport of fatty acids across the mitochondrial membrane, carnitine palmitoyl transferase 1 deficiency, had a fourfold increase in palmitoyl-CoA. Our results suggest that acyl-CoA esters in biological tissues are readily detectable using NCI mass spectrometry. This approach is significantly more sensitive than previous methods for the detection of these important metabolic intermediates, and may prove useful in the study of fatty acid oxidation in both normal and enzyme-deficient tissues.

The hypolipidemic agent fenofibrate, which is a peroxisome proliferator in some rodents in vivo, was studied in cultured hepatocytes for its metabolism and effects on enzymatic induction related to peroxisome proliferation so as to lead to a better understanding of the mechanisms involved in peroxisome proliferation. [14C]-Fenofibrate was completely metabolized within 24 hr by primary cultures of rat hepatocytes and the metabolic pattern corresponded to that found in vivo. The main products were fenofibric acid and its glucuronidated form. Carbonyl reduction of fenofibric acid also occurred. The metabolic pattern of [14C]fenofibric acid was nearly the same as that of fenofibrate. Fenofibrate, fenofibric acid, and its reduced metabolite all induced peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation activity (PCOA) in rat hepatocytes, whereas derivatives lacking the carboxyl group were nearly inactive. The known species differences with respect to sensitivity to peroxisome proliferators in vivo was mirrored in cultured cells because fenofibric acid did not induce peroxisomal PCOA in primary culture of guinea pig hepatocytes nor in the human hepatoma cell line HepG2. The mechanistic association between the induction of CYP4A1-catalyzed lauric acid omega-hydroxylase (LAH) activity and peroxisomal PCOA induction was investigated. Fenofibric acid concomitantly induced LAH activity and peroxisomal PCOA in rat hepatocytes. Specific inhibition of LAH activity (-52%) by 10-undecynoic acid partially prevented induction of peroxisomal PCOA (-32%). The putative role of dicarboxylic acids, the oxidation product of omega-hydroxymonocarboxylic acids, in PCOA induction was further substantiated by the observed induction of peroxisomal PCOA by 1-12-dodecanedioic acid. We conclude that (1) fenofibric acid is the possible proximate peroxisome proliferator of fenofibrate in rat hepatocytes, (2) cultured hepatocytes reflect in vivo sensitivity to fenofibrate with respect to peroxisome proliferation, and (3) there is some evidence that the catalytic activity of the CYP4A1 enzyme mediates, at least in part, peroxisomal PCOA induction.

A simple technique of quantitative cDNA-directed expression was devised by using a vaccinia virus system. The technique was applied to the cDNA expression of peroxisomal acyl-coenzyme A oxidase (AOX) and microsomal cytochromes P450 1A2, 2E1, and 3A4. Quantitative cDNA expression of all four enzymes tested was successfully achieved. This represents important progress for establishing the relationship between the amount of enzyme and its function in cells. The vaccinia virus system was also used to express AOX protein in specific cellular regions.

The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe3+ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H2O2 in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and alpha-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.

A sensitive and specific GTP-activated Ca2+ translocation process induces rapid Ca2+ movements within cells and appears to reflect G protein-induced membrane fusion or junctional communication between discrete subpopulations of Ca(2+)-pumping organelles (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Since fatty acylation can modify G protein action, modification of GTP-induced Ca2+ translocation by fatty acyl-CoA was investigated to throw light on the mechanism underlying Ca2+ transfer. Using permeabilized DDT1MF-2 smooth muscle cells, 2 microM palmitoyl-CoA completely blocked Ca2+ release activated by 20 microM GTP, while having no effect on inositol 1,4,5-trisphosphate-induced Ca2+ release. The IC50 (50% inhibitory concentration) for palmitoyl-CoA was 0.5 microM. Above 3 microM, palmitoyl-CoA inhibited Ca2+ accumulation. Fatty acyl chain length was important, C-13 to C-16 fatty acyl-CoA esters all fully blocking the action of GTP; the IC50 for myristoyl-CoA was also 0.5 microM. C-18 or larger acyl groups had diminished effectiveness as did C-8 or smaller acyl groups. Acetyl-CoA had no blocking effect. In contrast, 10 microM CoA itself blocked GTP-induced Ca2+ release. CoA required a free sulfhydryl group to block, desulfo-CoA having no effect. Removal of ATP by hexokinase and glucose prevented the action of CoA but not palmitoyl-CoA. The free sulfhydryl and ATP requirements indicated CoA was being acylated by endogenous fatty-acyl-CoA synthetase to be effective. The nonhydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, blocked the GTP effect identically to myristoyl- and palmitoyl-CoA (IC50 = 0.5 microM); thus, fatty acyl transfer is not required, indicating that blockade is due to a direct allosteric modification of a component of the GTP-activated process by acyl-CoA esters. Palmitoyl-CoA not only inhibited but completely reversed GTP-activated Ca2+ release, resulting in the released Ca2+ being taken back up into pools. In the presence of oxalate, GTP-activated Ca2+ transfer results in a substantial increase in Ca2+ accumulation; palmitoyl-CoA also completely reversed this effect resulting in rapid termination of Ca2+ uptake. This reversal provides strong evidence that GTP-activated Ca2+ translocation does not reflect a membrane fusion event. Instead, it likely represents formation of a reversible junction or pore between organelles which may be a required prefusion event.

The biosynthesis of glycosylphosphatidylinositol (GPI) in mammals and yeast involves a step not observed in trypanosomes. This reaction, which is the inositol acylation of glucosamine phosphatidylinositol (GlcN-PI), occurs as the third step in the biosynthetic pathway. In this study, conditions were developed to stimulate this reaction in vitro. The synthesis of the GlcN-PI(acyl) from either UDP-[6-3H]GlcNAc or [6-3H] GlcNAc-PI by murine lymphoma cell microsomes was greatly enhanced by the addition of either CoA or palmitoyl-CoA. Stimulation of this reaction was optimal with 1 microM of either compound and required that the precursor, GlcN-PI, be synthesized in the presence of GTP, a specific effector of the formation of this glycolipid. That GlcN-PI(acyl) was generated from GlcN-PI was established by pulse-chase analysis. Because no acyl-chain specificity for acyl-CoA stimulation of GlcN-PI(acyl) synthesis was found and attempts to demonstrate direct transfer of [3H]palmitate from [3H]palmitoyl-CoA to the third intermediate in GPI biosynthesis were unsuccessful, the possibility that free CoA was the activator of this reaction was considered. CoA-stimulated GlcN-PI acylation occurred in the absence of ATP, an essential cofactor for acyl-CoA synthesis, indicating that free CoA is the endogenous effector of the third step in mammalian GPI biosynthesis. This finding is consistent with this inositol acylation being catalyzed by a CoA-dependent transacylase. Mannose-containing GPI intermediates were synthesized in vitro when GDP-mannose was added in the presence of GTP and CoA. Therefore, when effectors of the initial reactions in GPI biosynthesis are included, later steps in this pathway can be studied in mammalian cell-free systems.

15N magnetic relaxation parameters T1, T2 and the nuclear Overhauser effect in the protein acyl-coenzyme A binding protein (ACBP) have been measured in the presence and absence of the ligand, palmitoyl-coenzyme A, in order to obtain information about local and global dynamical properties of the peptide backbone with and without the ligand bound in the binding site. The three-dimensional structures of acyl-coenzyme A binding protein are known for both states of the protein as determined from multidimensional heteronuclear NMR studies, and they have been shown to be essentially identical. However, the dynamics of the backbone is influenced by the presence of ligand in the binding site. The binding of ligand had significant and specific effects on the relaxation time T1 for many of the 15N in the peptide backbone, in particular those near residues with contacts to the ligand. Similarly, the nuclear Overhauser effect at 15N near such residues increased. There were no significant changes in the T2 relaxation. T1 values showing a significant decrease and NOEs increasing in regions close to the binding site when the ligand was bound suggest two modes of action on the dynamics of the protein when the ligand is binding. The reduced T1 indicates motion of lower amplitude in agreement with the structural constraints introduced by protein-ligand interactions. The increased NOEs may be a consequence of shorter time constants for dynamics of the atoms close to the binding site. The Lipari-Szabo model could not be satisfactorily applied to the entire set of experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)

Although migrating birds use stored triacylglycerol as their primary fuel for flight, they must retain sufficient stores of omega 6 and omega 3 fatty acids to sustain reproduction after the spring migration. Hepatic monoacylglycerol:acyl-coenzyme A acyltransferase (EC 2.3.1.22) (MGAT) activity is associated with physiological periods in which lipolysis and beta-oxidation are prominent, and it may also play a role in the selective retention of certain essential fatty acids. Therefore, we characterized MGAT activity in adipose tissue from the white-throated sparrow (Zonotrichia albicollis), a migratory bird. MGAT specific activity from adipose tissue and liver, respectively, was 22.2 +/- 7.27 and 0.79 +/- 0.35 nmol/min/mg of total particulate protein. Activity did not vary seasonally or between male and female birds. Specific activity increased 4.3-fold in the presence of 75 micrograms of phosphatidylcholine and phosphatidylserine (1:1, w/w). MGAT acylated sn-1(3)-monooleoylglycerol, sn-2-monooleylglycerol ether and sn-1(3)-monooleylglycerol ether at 7.5, 5.7 and 1.7%, respectively, of the rate observed with sn-2-monooleoylglycerol. An initial lag phase observed at low concentrations of palmitoyl-CoA was corrected by adding 2 mM MgCl2, Mg(NO3)2 or CaCl2, suggesting a requirement for divalent cations. MGAT acylated sn-2-monolinolenoylglycerol and sn-2-monolinoleoylglycerol in preference to sn-2-monooleoylglycerol. Specificity of MGAT for sn-2-monoacylglycerols and the probable enhanced affinity fo sn-2-monoacylglycerols of specific acyl chains may allow selected omega 6 and omega 3 fatty acids to be retained within the adipocyte, while nonessential fatty acids are released for beta-oxidation in flight muscles.

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low microM concentrations of octanoyl-CoA. However, even a large excess (500 microM) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low microM concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above affect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolism in vivo.

The contents of peroxisomal fatty acid beta-oxidation enzymes in three rat hepatoma cell lines, i.e., H4IIEC3 (H4), N1S1, and McA-RH7777 (H7), were measured by immunoblot analysis, and a significant difference in acyl-coenzyme A oxidase (AOX) content became evident. These cell lines were respectively infected with a recombinant virus to express significant amounts of AOX protein. The expressed AOX mainly localized in organelle, supposing peroxisomes, and was catalytically active. The cDNA-expression in H4, N1S1, and H7 cells enhanced 2.6-, 2.2-, and 1.0-fold beta-oxidation activity of lignoceric acid, respectively. The enhancement in H4 and N1S1 cells suggests that AOX is a rate-limiting enzyme in the very-long-chain fatty acid beta-oxidation system, in these cell lines.

Two tandem cysteine residues in the carboxyl-terminal region of rhodopsin have been shown to be covalently linked to palmitate via thioester bonds (Ovchinnikov, Y. A., et al. (1988) FEBS Lett. 230, 1-5). We have synthesized a fluorescent analogue of palmitoyl coenzyme A (16-(9-anthroyloxy)hexadecanoyl coenzyme A ester) and incorporated the fluorescent derivative of palmitate into the protein in high yield (> 40%) through pretreatment of bovine rod outer segments with 1 M hydroxylamine and subsequent incubation with the fluorescent label. Covalent incorporation of label into protein was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic digestion of labeled rhodopsin in the disc membrane with papain and thermolysin verified the C-terminal location of the label. Treatment of SDS-solubilized, labeled rod outer segments with 10% beta-mercaptoethanol provided evidence that partial depalmitoylation may induce the formation of rhodopsin aggregates. Labeled, unbleached rhodopsin was purified by chromatography over hydroxyapatite and concanavalin A-agarose and reconstituted into dimyristoylphosphatidylcholine vesicles. SDS gels of the rhodopsin vesicle preparation verified that all unbound fluorescent label had been removed and that the thioester bond linking probe to protein was not labile.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. In this report, a simple and rapid purification as well as the properties of this enzyme from bovine spleen is described. Using combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow, phenyl-Sepharose CL-4B, DEAE-Sepharose CL-6B, and Superose 12 (HR/30) gel filtration fast protein liquid chromatography, the enzyme was purified 1475-fold with a high yield. Under native conditions, the enzyme exhibited an apparent molecular mass of 58 kDa, whereas under denaturing conditions the enzyme represented an apparent molecular mass of 50 kDa, suggesting that spleen NMT is a monomeric protein. The NMT activity could be greatly activated to severalfold with the use of Tris-HCl buffer. Kinetic properties indicated that spleen NMT had an apparent low Km for pp60src and myristoylated alanine-rich C kinase substrate as compared with cAMP-dependent protein kinase and the M2 gene segment of reovirus type 3-derived peptides. Bovine spleen NMT was potently inhibited in a concentration-dependent manner by NIP71 (a bovine brain NMT inhibitory protein) with a half-maximal inhibition of 0.816 microgram/ml. Results of this study along with the existing knowledge on NMT indicate that the activity of enzyme resides in a single polypeptide chain of molecular mass between 50 and 68 kDa.

The acylation of membrane phospholipids in isolated bovine rod outer segments (ROS) by exogenous, radiolabeled palmitic acid (16:0) was assayed, using [14C]palmitoyl-coenzyme A (16:0-CoA), [3H]16:0 alone and [3H]16:0 with added ATP and CoA. Both [14C]16:0-CoA and [3H]16:0 with added ATP and CoA are incorporated predominantly into phosphatidylcholine (PC), whereas [3H]16:0 without added ATP and CoA is incorporated almost exclusively into phosphatidylethanolamine (PE). Without added ATP and CoA in the incubation medium, [3H]16:0 is esterified predominantly to the 2 position of PE, but with added ATP and CoA, it is esterified to both the 1 and 2 positions; the rates of esterification of [3H]16:0 to the 2 position of PE under these two conditions are similar. These results suggest that 16:0 is esterified to the 2 position of PE of ROS membranes by an acyl transferase mechanism that does not utilize an acyl-CoA intermediate. The product of this reaction is likely to be a molecular species of PE with 16:0 at the 2 position and a polyunsaturated fatty acid at the 1 position.

We have used microsomes prepared from murine lymphoma cell lines to investigate the individual reactions by which glycosylphosphatidylinositol (GPI) is synthesized in mammalian cells. Previously, GTP was found to specifically stimulate the second reaction in the pathway, the deacetylation of GlcNAc-PI to GlcN-PI. An additional GPI precursor was detected in incubations with GTP and was found to be GlcN-PI(acyl), the glycolipid proposed to be the third intermediate in mammalian GPI biosynthesis. Investigation into the factors that affect the formation of GlcN-PI(acyl) revealed that, in the presence of GTP, the addition of either CoA or palmitoyl-CoA to the incubation greatly enhanced the amount of this product made. CoA stimulation of this reaction persisted even when ATP was depleted and no formation of acyl-CoA was possible, indicating that the free CoA rather than an acyl-CoA is the actual effector of GlcN-PI acylation. Therefore, we propose that the third reaction in mammalian GPI biosynthesis is catalyzed by a CoA-dependent transacylase rather than an acyl-CoA acyltransferase.

Triacylglycerol is synthesized from the precursors sn-1,2-diacylglycerol and and palmitoyl-CoA in a reaction catalyzed by the microsomal enzyme diacylglycerol acyltransferase (EC 2.3.1.20). Isolated 3T3-L1 adipocyte microsomal vesicles from cells pulse-labeled with L-[35S]methionine were found to release microsomal proteins into a low density form during the synthesis of triacylglycerol. The proteins released, which represent a subset of those present in the labeled microsomes, include a 62-kDa protein found in high concentration in mature fat droplets. The formation of the triacylglycerol-protein complexes was dependent on time and temperature, was not stimulated by cytosol, and required ATP as well as diacylglycerol and palmitoyl-CoA. Only nucleoside triphosphates and not non-hydrolyzable analogues could replace ATP in the reaction. Unlike the enzyme reaction that measures the synthesis of triacylglycerol, the formation of low density membrane is thus dependent on ATP hydrolysis as well as enzyme substrates. The newly formed, low density particles are selectively enriched in triacylglycerol synthesized during the reaction as well as that synthesized prior to the reaction. The cell-free system described thus appears to represent an early adipogenic event leading to the lipid vacuoles found in mature adipocytes.

Most effects of the peroxisome proliferator clofibrate on rat liver are marginal or absent in selenium (Se) deficiency. The purpose of the present study was to determine whether the uptake or distribution of clofibrate is altered by Se deficiency. Rats were fed a Se-adequate or -deficient diet for 10-11 weeks and then these same diets with 0.5% (w/w) clofibric acid (the direct acting hydrolysis product of clofibrate) or 0.02% (w/w) perfluorooctanoic acid (PFOA) for 10 days. Other groups of rats received radiolabeled clofibrate by intubation. Clofibric acid was an ineffective as clofibrate in producing effects (i.e. decreased body weight gain, increases in liver somatic index and protein content of the mitochondrial fraction, and increased activities of catalase and peroxisomal fatty acid beta-oxidation) in the liver of Se-deficient rats. Microsomal omega-hydroxylation was, however, equally induced in both dietary groups. In contrast to clofibric acid, the biological effects of PFOA were not affected by Se status. Furthermore, neither the tissue distribution (plasma, liver and kidney) nor the urinary excretion of 14C was affected by Se deficiency. These results demonstrate that the hydrolysis of clofibrate to clofibric acid is not impaired in the Se-deficient rat. In addition, the involvement of Se in the effects of peroxisome proliferators differs for different members of this structurally heterogeneous group of compounds. It is concluded that the Se-deficient rat may provide valuable information concerning the biochemical mechanism(s) underlying peroxisome proliferation.

A carboxylesterase containing long-chain acyl-CoA hydrolase activity was purified to apparent homogeneity from rat liver microsomes. Palmitoyl-CoA was the most preferred substrate, followed by stearoyl-CoA and oleoyl-CoA. Arachidonoyl-CoA, linoleoyl-CoA and acetyl-CoA were not hydrolysed by the enzyme. The purified enzyme had no activity on the hydrolysis of phospholipids and neutral lipids. The molecular mass of the enzyme was found to be 56 kDa by SDS/PAGE and 64 kDa by gel-filtration chromatography. On isoelectric focusing, the purified enzyme behaved like the ES-4 type, with a pI of 6.15. Determination of the amino acid sequence revealed that its N-terminal sequence is 100% homologous with the only other known N-terminal sequence for a rat carboxylesterase isoenzyme (ES-10). Enzyme activity was inhibited by lysophosphatidic acid and activated by lysophosphatidylcholine. The modulation of enzyme activity by these lysophospholipids might represent a plausible mechanism for the physiological control of acyl-CoA concentrations.

Palmitoyl-coenzyme A (palmitoyl-CoA) hydrolase was obtained from rat kidney in an electrophoretically homogeneous form. The enzyme associated with carboxylesterase activity was purified by acetone precipitation of microsomes, followed by successive chromatographies on DEAE-cellulose, phenyl-Sepharose, and Sephadex G-100 gel. The two activities in rat kidney were associated as judged by their co-elution profiles, co-purification at different steps, co-precipitation by an antibody raised against the purified enzyme, and identical profiles of inhibition by diisopropylfluorophosphate. The enzyme catalyzed the hydrolysis of long- and medium-chain acyl-CoA, but not short-chain acyl-CoA. The N-terminal amino acid sequence of the first 27 residues of the purified enzyme was 80% identical with that of the carboxylesterase from rat adipose tissue. Using a polyclonal rabbit antibody against the rat kidney palmitoyl-CoA hydrolase, the enzyme was demonstrated in liver but not in adipose tissue. The antibody reacted with the carboxylesterase(s) (pI 6.3 and pI 6.6) in rat liver microsomes. The antibody removed the palmitoyl-CoA hydrolase in kidney (75%) and liver (68%). The antibody also removed the monoolein hydrolase in kidney (77%) and liver (61%). These results suggest that carboxylesterase contributes to the hydrolysis of long-chain acyl-CoA and monoglyceride in kidney and liver.

Male Wistar albino rats were treated for a 7 day period with equimolar doses of the trimer and tetramer oligomers of chlorotrifluoroethylene (CTFE), resulting in significant hepatomegaly for both compounds. In addition, both trimer and tetramer significantly induced the peroxisomal beta-oxidation of fatty acids as assessed by increases in palmitoyl-coenzyme A (CoA) oxidation, thus confirming these oligomers as peroxisome proliferators. Consistent with these conclusions, both trimer and tetramer increased the hydroxylation of lauric acid indicating that the CTFEs were inducers of the CYP4A subfamily, a conclusion further supported by substantial increases in the steady-state levels of the cognate CYP4A1 mRNA as determined by northern blotting. The liver appeared to be more susceptible to induction than the kidney and the CTFE tetramer was more potent than the trimer. These results are discussed with respect to both the differential hepatotoxicity, and biotransformation/disposition of the two polyhalogenated oligomers.

1. Male mice were fed a diet containing perfluoro fatty acids of varying chain length (i.e. perfluoroacetic, -butyric, -octanoic and -decanoic acids) at different doses (0.02 or 0.1% w/w of diet) for different periods of time (2-10 days), and effects on liver weight, hepatic mitochondrial protein and hepatic peroxisomal palmitoyl-CoA oxidation, lauroyl-CoA oxidase and catalase were monitored. 2. The greatest effects were obtained with perfluoro-octanoic and perfluoro decanoic acids, while perfluoro acetic acid was inactive. The effects with 0.02% w/w of diet perfluoro-octanoic acid were at least as great as those observed with 0.1%. A more detailed dose-response investigation focused on perfluoro-octanoic acid revealed that maximal effects with this substance could be obtained with a dietary dose of 0.01% for 10 days and that significant changes were also observed with 0.001%. 3. Maximal effects with 0.02% w/w of diet perfluoro-octanoic acid were attained after 6-10 days of feeding. 4. As with other peroxisome proliferators, perfluoro fatty acids increase mouse hepatic peroxisomal fatty acid beta-oxidation more extensively than they increase catalase, thus increasing hepatic oxidative stress. 5. As with other peroxisome proliferators, perfluoro fatty acids increase mouse liver mitochondrial protein. This effect is due primarily to a redistribution of mitochondria from the nuclear to the mitochondrial fraction, caused by an apparent decrease in the mean size of hepatic mitochondria after treatment.

Hepatic beta-oxidation is characterized in a marine teleost, Myoxocephalus octodecimspinosus, to determine mitochondrial and peroxisomal substrate selectivity as well as metabolic partitioning. Substrate selectivity is broad for peroxisomal beta-oxidation. Acyl CoA oxidase activities, with all unsaturated substrates measured, are at least 35% of activity with palmitoyl CoA (16:0), a saturated substrate. Mitochondrial selectivities are more pronounced. Carnitine palmitoyltransferase activity with a monounsaturate, palmitoleoyl CoA (16:1), is nearly 40% greater than activity with palmitoyl CoA, whereas activities with two polyunsaturates are < 10% of activity with the saturate. The presence of polyunsaturated acyl CoA esters inhibits up to 70% the oxidation of palmitoyl CoA by intact peroxisomes. Acyl CoA hydrolase activity is localized to peroxisomal fractions prepared by density-gradient centrifugation. Hydrolytic activity in these fractions is nearly twofold the activity of beta-oxidation. Estimates for metabolic partitioning suggest that at least 50% of hepatic beta-oxidation may be initiated by the peroxisomal compartment.

Esterification of all-trans-retinol is a key reaction of the vertebrate visual cycle, since it produces an insoluble, relatively non-toxic, form of the vitamin for storage and supplies substrate for the isomerization reaction. CoA-dependent and -independent pathways have been described for retinol esterification in retinal pigment epithelium (RPE). The CoA-independent reaction, catalysed by lecithin:retinol acyltransferase (LRAT) was examined in more detail in this study. Addition of retinol to RPE microsomes results in a burst of retinyl ester synthesis, followed by a rapid apparent cessation of the reaction. However, [3H]retinol, added when retinyl ester synthesis has apparently ceased, is rapidly incorporated into retinyl ester without a net increase in the amount of ester. The specific radioactivities of [3H]retinol and [3H]retinyl ester reach the same value. [14C]Palmitate from palmitoyl-CoA is incorporated into preexisting retinyl ester in the absence of net ester synthesis, too. These exchange reactions suggest that the reaction has reached equilibrium at the plateau of the progress curve and that only the accumulation of retinyl ester, and not its synthesis, has stopped during this phase of the reaction. Studies with geometrical isomers of retinol revealed that the rate of exchange of all-trans-retinol with all-trans-retinyl esters was about 6 times more rapid than exchange of 11-cis-retinol with 11-cis-retinyl ester. This is the first demonstration of the reversibility of LRAT and the first example of stereospecificity of retinyl ester synthesis in the visual system. Reversal of the LRAT reaction could contribute to the mobilization of 11-cis-retinol from 11-cis-retinyl ester pools.