Subcellular distribution of acetyl-coenzyme A carboxylase in mesophyll cells of barley and sorghum leaves

The subcellular distribution of acetyl-CoA carboxylase [acetyl-CoA-carbon dioxide ligase (ADP-forming), EC 6.4.1.2] was determined in mesophyll protoplasts isolation from barley, a C3 plant, and sorghum, a C4 plant. In both species, all of the mesophyll acetyl-CoA carboxylase was demonstrated to be chloroplastic. In barley leaves and mesophyll protoplasts, a single biotinyl protein of 60,000 Da was identified by a modified Western-blotting procedure. The subcellular distribution of this biotinyl protein was identical to that found for acetyl-CoA carboxylase. These results are discussed in relation to the compartmentation of reactions requiring malonyl-CoA as a substrate.     

Pyruvate carboxylase in lactating rat and rabbit mammary gland

1. Pyruvate carboxylase [pyruvate-carbon dioxide ligase (ADP), EC 6.4.1.1] was found in cell-free preparations of lactating rat and rabbit mammary glands, and optimum assay conditions for this enzyme were determined. 2. Subcellular-fractionation studies with marker enzymes showed pyruvate carboxylase to be distributed between the mitochondrial and soluble fractions of lactating rat mammary gland. Evidence is presented that the soluble enzyme is not an artifact due to mitochondrial damage. 3. In contrast, pyruvate carboxylase in lactating rabbit mammary gland is confined to the mitochondrial fraction. 4. The final product of pyruvate carboxylase action in the mitochondrial and particle-free supernatant fractions of lactating rat mammary gland was shown to be citrate. 5. The effects of freeze-drying, ultrasonic treatment and freezing-and-thawing on the specific activity of mitochondrial pyruvate carboxylase were investigated.     

Fatty-acid synthesis in lactating-goat mammary gland. 1. Medium-chain fatty-acid synthesis.

Tissue slices from lactating goat-mammary gland synthesized short (C4:0 and C6:0), medium (C8:0 and C10:0) and long-chain (C12:0 to C16:0) fatty acids in proportions similar to that found in goat milk fat. In contrast, the particle-free supernatant fraction and the purified fatty acid synthetase from this tissue synthesized predominantly short-chain and long-chain fatty acids. Terminating acyl-thioesterases of low molecular weight could not be detected in the particle-free supernatant. Addition of the microsomal fraction to the particle-free supernatant induced the synthesis of medium-chain fatty acids in proportions which were similar to those found in goat milk fat.     

Fatty acids bound to unilamellar lipid vesicles as substrates for microsomal acyl-CoA ligase

Palmitate incorporated into single-layered vesicles of phosphatidylcholine was used as a substrate for palmitoyl coenzyme A ligase (palmitoyl-CoA ligase) in microsomes from rat liver. This was done in order to avoid the use of detergents for dispersal of the water-insoluble palmitate and the possibility of precipitating palmitate added to the aqueous assay as a salt suspension. The activity of the ligase measured when palmitate was added to assays as a component of phospholipid vesicles was 10-40-fold greater vs. activities reported in the literature using other methods for adding fatty acids to the assay system. Phospholipids, however, had no direct effect on the activity of palmitoyl-CoA ligase. The data indicate, therefore, that the activity of this enzyme has been underestimated because of the manner in which fatty acid was added to the assay, which has a significant effect on the activity of the ligase. It is shown too that the rate of spontaneous transfer of palmitate from unilamellar vesicles of phosphatidylcholine to microsomes via a hydrated intermediate is far more rapid than the inherent catalytic activity of the fatty acyl-CoA ligase. The data also suggest that the membrane-associated pool of fatty acid and not fatty acid in the aqueous phase of the assay is the pool of substrate interacting with the ligase.     

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.     

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue.     

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.     

Triglyceride synthesis by small-intestinal epithelium of the pig, sheep and chicken

1. A comparative study was made of triglyceride synthesis by the intestinal epithelium of pigs, sheep and chickens. In pig and chicken tissue both the glycerol 3-phosphate and the monoglyceride pathway of triglyceride synthesis were operative, but the former pathway predominated in sheep tissue. 2. The fatty acid specificity of the glycerol 3-phosphate pathway was studied in pig and sheep total-homogenate preparations. Maximum incorporation was obtained with myristic acid and palmitic acid under optimum conditions for each fatty acid. Lauric acid, myristic acid, oleic acid, linoleic acid and linolenic acid were inhibitory at concentrations above their optimum, but octanoic acid, decanoic acid, palmitic acid and stearic acid did not show this effect. 3. Subcellular fractionation located the glycerol 3-phosphate and monoglyceride pathways of triglyceride synthesis in the microsomes in all instances. Phosphatidate phosphohydrolase was associated with both the microsomes and the particle-free supernatant. 4. Glycerol 1-mono-oleate was incorporated into triglycerides to a greater extent than glycerol 1-mono-palmitate or glycerol 1-monostearate by microsomal preparations from pig and chicken. 5. A lipase specific for monoglycerides was detected in the particle-free supernatant of all the species examined.     

Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.     

Adrenoleukodystrophy. The chain shortening of erucic acid (22:1(n-9)) and adrenic acid (22:4(n-6)) is deficient in neonatal adrenoleukodystrophy and normal in X-linked adrenoleukodistrophy skin fibroblasts

The metabolism of long chain unsaturated fatty acids was studied in cultured fibroblasts from patients with X-linked adrenoleukodystrophy (ALD) and with neonatal ALD. By using [14-14C] erucic acid (22:1(n-9)) as substrate it was shown that the peroxisomal beta-oxidation, measured as chain shortening, was impaired in cells from patients with neonatal ALD. The beta-oxidation of adrenic acid (22:4(n-6)), measured as acid-soluble products, was also reduced in the neonatal ALD cells. The peroxisomal beta-oxidation of [14-14C]erucic acid (22:1(n-9)) and [2-14C]adrenic acid (22:4(n-6)) was normal in cells from X-ALD patients. The beta-oxidation, esterification and chain elongation of [1-14C]arachidonic acid (20:4(n-6)) and [1-14C]eicosapentaenoic acid (20:5(n-3)) was normal in both X-linked ALD and in neonatal ALD. Previous studies suggest that the activation of very long chain fatty acids by a lignoceryl (24:0)-CoA ligase is deficient in X-linked ALD, while the peroxisomal beta-oxidation enzymes are deficient in neonatal ALD. The present results suggest that the peroxisomal very long-chain acyl-CoA ligase is not required for activation of unsaturated C20 and C22 fatty acids and that these fatty acids can be efficiently activated by the long chain acyl-(palmityl)-CoA ligase.     

Preservation of lipid reesterifying enzymes in the microsomal fraction of the rat jejunum

Studies were performed on methods of storage of rat jejunal tissue that would preserve activities of the lipid reesterifying enzymes, acyl CoA:monoglyceride acyltransferase and fatty acid CoA ligase. Storage at -80 degrees C of microsomes prepared from jejunal mucosa or storage of lyophilized microsomes at -20 degrees C was shown to preserve acyl CoA:monoglyceride acyltransferase very well for a matter of weeks. Preservation of fatty acid CoA ligase activity was adequate with either method, but results were not as good as for the transacylase enzyme.     

Fatty acid biosynthesis in the peripheral nervous system of normal and Trembler mice

De novo fatty acid biosynthesis was demonstrated in a particle-free supernatant from normal and Trembler mouse sciatic nerves. In both systems, it required acetyl-CoA and malonyl-CoA, and led chiefly to the formation of free palmitic acid. No palmitoyl-CoA formation was detected. The ability of the cell-free extract of the mutant to form palmitic acid in vitro was greater than that of the control extracts when the results were expressed as the total activity per 100 mg of freshly excised sciatic nerves.     

The synthesis of medium-chain fatty acids by lactating-rabbit mammary gland studied in vitro (Short Communication)

The proportion of C(8:0) and C(10:0) fatty acids synthesized by the microsomal plus particle-free supernatant fraction from lactating rabbit mammary gland is enhanced at high protein concentrations. This fraction appears to contain a soluble high-molecular-weight factor that modifies the specificity of the fatty acid synthetase complex for termination of the growing acyl chain.     

Millipore filter assay for long-chain fatty acid:CoASH ligase activity using 3H-labeled coenzyme A.

A novel radiochemical assay for long-chain fatty acid:CoASH ligase activity (AMP) (EC 6.2.1.3) has been developed based on the conversion of [3H]CoASH to long-chain fatty acyl CoA. Fatty acyl [3H]CoA was quantitatively retained on Millipore filters upon filtration of the acidified reaction mixture under conditions where the [3H]CoASH was not retained. The assay was developed using microsomes derived from isolated fat cells as the source of fatty acid:CoASH ligase activity. The assay performed at 25 degrees C for 10 min was linear with added microsomal protein up to 7 mug. The assay was linear with time up to 24 min when 1 mug of protein was employed. Fatty acid:CoASH ligase activity was strongly dependent on ATP and magnesium, was stimulated by Triton WR-1339, and was two- to fivefold dependent on added fatty acid. The filter assay is easier than existing assays based on incorporation of labeled fatty acid and is equally sensitive.     

Substrate specificity of fatty-acyl-CoA ligase in liver microsomes

The substrate specificity of fatty-acyl-CoA ligase in liver microsomes has been studied in a system in which fatty acids are present initially as complexes with unilamellar vesicles of phosphatidylcholine. The latter were prepared by cosonication of phospholipids and different fatty acids. As compared with previous studies of the enzyme the activity of acyl-CoA ligase is several-fold higher for assays carried out with fatty acid substrates added as components of a bilayer. This was true for all fatty acids studied. Also as compared with data reported previously in the literature there was a systematic relationship between the structure of fatty acids, activity at V_max for synthesis of acyl-CoA and avidity of binding to the ligase. Activity at V_max was greatest for lauric acid and decreased with increasing chain length. The apparent avidity of enzyme for fatty acids was greatest for octanoic acid and decreased as chain length increased.     

Possible association of segregated lipid domains of Mycoplasma gallisepticum membranes with cell resistance to osmotic lysis.

Freeze-fracturing of cholesterol-rich Mycoplasma gallisepticum membranes from cells grown in a medium containing horse serum revealed particle-free patches. The patches appeared in cells quenched from either 4 or 37 degrees C. Particle-free patches also occurred in membranes of cells grown in a serum-free medium supplemented with egg-phosphatidylcholine but not in membranes of cells grown with dioleoylphosphatidylcholine. The appearance of particle-free patches was attributed to the presence of disaturated phosphatidylcholine (PC) molecules in M. gallisepticum membranes, which were synthesized by the insertion of a saturated fatty acid at position 2 of lysophosphatidylcholine derived from exogenous PC present in the growth medium. Consequences of the synthesis of the disaturated PC also included a decrease in osmotic fragility and the ability of the cells to be permeated by K+. Electron paramagnetic resonance and fluorescence polarization measurements revealed that the fluidity of the lipid domain in the protein-rich M. gallisepticum membranes was almost identical to that of an aqueous dispersion of M. gallisepticum membrane lipids. Furthermore, the electron paramagnetic resonance spectra of the membranes were single-component spectra showing no indication of immobilized regions. The possibility that the osmotic resistance of M. gallisepticum cells is associated with the particle-free patches rather than with a restricted membrane fluidity caused by membrane proteins is discussed.     

Unsaturated fatty acid requirement inEscherichia coli: Mechanism of palmitate-induced inhibition of growth of strain WN1

Summary The minimum requirement for unsaturated fatty acids was investigated inE. coli using a mutant impaired in the synthesis of vaccenic acid. Exogenously supplied palmitic acid was incorporated by this mutant which led to a reduction in the proportion of cellular unsaturated fatty acids. Growth was impaired as the level of saturated fatty acids approached 76% at 37°C and 60% at 30°C. The basis of this growth inhibition was investigated. Most transport systems and enzymes examined remained active in palmitate-grown cells although the specific activities of glutamate uptake and succinic dehydrogenase were depressed 50%. Fluorescent probes of membrane organization indicated that fluidity decreased with palmitate incorportation. Temperature scans with parinaric acid indicated that rigid lipid domains exist in palmitategrown cells at their respective growth temperature. Freeze-fracture electron microscopy confirmed the presence of phase separations (particle-free areas) in palmitate-grown cells held at their growth temperature prior to quenching. The extent of this separation into particle-free and particle-enriched domains was equivalent to that induced by a shift to 0°C in control cells. The incorporation of palmitate increased nucleotide leakage over threefold. The cytoplasmic enzyme -galactosidase was released into the surrounding medium as the concentration of unsaturated fatty acid approached the minimum for a particular growth temperature. Lysis was observed as a decrease in turbidity when cells which had been grown with palmitate were shifted to a lower growth temperature. From these results we propose that leakage and partial lysis are the major factors contributing to the apparent decrease in growth rate caused by the excessive incorporation of palmitate. Further, we propose that membrane integrity may determine the minimum requirement for unsaturated fatty acids inE. coli rather than a specific effect on membrane transport and/or membrane-bound enzymes.     

Citrate and the conversion of carbohydrate into fat. The regulation of fatty acid synthesis by rat liver extracts

1. The rate of fatty acid synthesis by particle-free extracts prepared from rat liver is increased greatly if the enzyme system is first activated with citrate. 2. The extent of the activation depends on the citrate concentration and on the time of activation in an interdependent manner. 3. Citrate activation is strongly dependent on temperature. 4. Tricarballylate can replace citrate as an activator, but its presence in the assay inhibits fatty acid synthesis. 5. Mg(2+) ions can replace citrate in the activation but not in the complete reaction system. 6. ATP prevents the activating effect of citrate and Mg(2+) ions. 7. The rate of fatty acid synthesis is increased by palmitoyl-dl-carnitine. This type of activation, additional to that caused by citrate, is rapid and does not depend on prior incubation. 8. Inhibition of fatty acid synthesis by palmitoyl-CoA can be prevented by palmitoyl-dl-carnitine or by increasing the concentration of protein.     

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Abstract: We measured long-chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl-CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.     

Characterization of rat brain microsomal acyl-coenzyme A ligases: different enzymes for the synthesis of palmitoyl-coenzyme A and lignoceroyl-coenzyme A

Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes.