Rat Liver Fatty Acid Synthetase Research Articles

Stoichiometry of substrate binding to rat liver fatty acid synthetase.

Two rat liver fatty acid synthetase preparations, containing 1.6 and 2.0 mol of 4'-phosphopantetheine/mol of synthetase, showed specific activity of 2006 and 2140 nmol of NADPH oxidized/min per mg of protein respectively. The two synthetase preparations could be loaded with either 3.3-4.4 mol of [1-14] acetate or 2.9-3.7 mol of [2-14C]malonate, by incubation with either [1-14C] acetyl-CoA or [2-14C]malonyl-CoA. The 4'-phosphopantetheine site could be more than 90% saturated and the serine site about 80% saturated with malonate derived from malonyl-CoA. However, with acetyl-CoA as substrate, binding at both the 4'-phosphopantetheine and cysteine thiol sites did not reach saturation. We interpret these results to indicate that, whereas the equilibrium constant for transfer of substrates between the serine loading site and the 4'-phosphopantetheine site is close to unity, that for transfer of acetyl moieties between the 4'-phosphopantetheine and cysteine sites favours formation of the 4'-phosphopantetheine thioester. Thus, despite the apparent sub-stoichiometric binding of acetate, the results are consistent with a functionally symmetrical model for the fatty acid synthetase which permits simultaneous substrate binding at two separate active centres.

Cloning of DNA complementary to rat liver fatty acid synthetase mRNA.

Clones, containing DNA complementary (cDNA) to rat liver fatty acid synthetase mRNA, were constructed and identified. cDNA of these clones was then used as a probe to quantify mRNA. The cDNA was synthesized to partially purified rat liver fatty acid synthetase mRNA. Double-stranded cDNA was then prepared and inserted into the PstI site of pBR322 using oligo(dG) X oligo(dC) tailing. Initial selection of the clones was by differential colony hybridization employing [32P]cDNA synthesized from poly(A)-rich mRNA, enriched and non-enriched in fatty acid synthetase mRNA, as probes. Plasmids, containing specific sequences complementary to the fatty acid synthetase mRNA, were identified by hybrid-arrest translation. Cloned cDNA inserts ranged from 300 to 1400 base pairs. Cloned cDNA was employed to probe for mRNA in hybridizations via the dot-blot method. These studies demonstrated an increase in fatty acid synthetase mRNA during dietary induction, which suggests that regulation may involve changes in transcription or changes in post-transcriptional processing of the mRNA.

The involvement of a lysine residue at the active site of the enoyl reductase of pigeon liver fatty acid synthetase

The reaction of pyridoxal-5′-phosphate with lysine residues of pigeon and rat liver fatty acid synthetases has been investigated. The enzyme is reversibly inactivated by this compound inasmuch as overall and enoyl reductase activities are lost as a function of time. β-Ketoacyl reductase activity and other component activities of the enzyme are not affected. Spectrophotometric assays, as well as amino acid analysis of the acid hydrolysate of NaB 3 H 4 -reduced enzyme-pyridoxal phosphate complex, have established that the inactivation of the enzyme is due to Schiff base formation with e-amino groups of lysine residues that are essential for the enoyl reductase component activity of fatty acid synthetases.

Comparative studies on the kinetic parameters and product analyses of chicken and rat liver and yeast fatty acid synthetase

1. 1. Comparative kinetics and product analyses of chicken and rat liver and yeast fatty acid synthetase. 2. 2. V max' s for the three enzymes studied decrease with increasing primer chain-length, while K m' s (except for yeast) increases. 3. 3. Palmitate is the main product (∼90%) of the rat synthetase whereas palmitate (60%) and stearate (40%) are the products of chicken and yeast enzymes. Increasing the primary chain length does not alter palmitate synthesis by rat and chicken enzymes but increases stearate synthesis by the yeast synthetase. 4. 4. The three synthetases could not synthesize fatty acids in the absence of primer acetyl-CoA suggesting that malonyl-CoA decarboxylase is not a component.

Demonstration of the occurrence of inactive fatty acid synthetase in rat liver by immunotitration and its in vitro partial activation.

Direct immunotitrations of rat liver fatty acid synthetase in crude tissue homogenates with monospecific rabbit anti-rat liver fatty acid synthetase antibody enabled us to make a comparison of fatty acid synthetase protein and activity (percentage of maximal activity) as a function of the nutritional state in normal, diabetic, and insulin- and glucagon-insulin treated animals. Previous results, in which large changes in fatty acid synthetase activity were related to protein synthesis and degradation rather than to enzyme activation, were confirmed. It was also shown that fatty acid synthetase activation does not occur immediately on synthesis but follows the synthesis of fatty acid synthetase protein. In order to characterize the enzymatically inactive protein found on immunotitration and to develop an in vitro system for fatty acid synthetase activation, conditions were sought to obtain large amounts of fatty acid synthetase protein free from, or low in, activity. It was found that treatment of hypophysectomized rats with triiodothyronine meets these requirements, yielding milligram quantities of inactive fatty acid synthetase protein with less than 2% of maximal activity. A part of the inactive fatty acid synthetase was found to be the apoenzyme as indicated by beta-ketoreductase and thioesterase activities, by its ability to incorporate label from [G3H]CoA, and by its partial in vitro activation, which led to an increase in overall synthetase activity in crude and partially purified cell-free systems. The components required for activation include magnesium ion and a transferase fraction prepared from livers of 48-h fasted, 12-h refed rats.

Purification and crystallization of rat liver fatty acid synthetase

A purification procedure for rat liver fatty acid synthetase has been developed using polyethylene glycol. This procedure results in high yields of the enzyme which is essentially free of endogenous proteolytic nicking and also free of any contaminating proteases. The fatty acid synthetase obtained has a specific activity range of 1.8–2.1 measured at 25 °C and is stable at 4 °C for a few weeks and indefinitely when frozen. Approximately 1 mg of enzyme can be obtained per gram of induced rat liver. The enzyme is pure as determined by sodium dodecyl sulfate-gel electrophoresis, sedimentation velocity, and immunoelectrophoresis. The first crystallization of rat liver fatty acid synthetase is also reported.

The coenzyme A requirement of mammalian fatty acid synthetase: Evidence for involvement in the elongation of acyl-enzyme thioesters

We have confirmed that coenzyme A is required for rat fatty acid synthetase activity (T. C. Linn, M. J. Stark, and P. A. Srere, 1980, J. Biol. Chem. 255, 1388–1392) . When rat liver or mammary gland fatty acid synthetase was assayed in the presence of a CoA-scavenging system such as ATP citrate lyase, almost complete inhibition of fatty acid synthesis was observed. The inhibition was reversed by addition of CoA or pantetheine, but not by addition of N-acetylcysteamine or other thiols. In the absence of CoA, the rate of elongation of acyl moieties on both native fatty acid synthetase and fatty acid synthetase lacking the chain-terminating thioesterase I component (trypsinized fatty acid synthetase) was reduced 100-fold. All of the palmitate synthesized slowly by the CoA-depleted native multienzyme was released, by the thioesterase I component, as the free fatty acid; only shorter-chainlength acyl moieties remained bound to the enzyme. The acyl- S-multienzyme thioesters formed by the trypsinized fatty acid synthetase in the absence of CoA contained saturated moieties of chain length C 6-C 16; addition of CoA promoted elongation of the acyl- S-multienzyme thioesters without release from the enzyme. The transfer of acetyl and malonyl moieties from CoA to the multienzyme, the reduction of S-acetoacetyl- N-acetylcysteamine and S-crotonyl- N-acetylcysteamine, and the dehydration of S-β-hydroxybutyryl- N-acetylcysteamine, reactions catalyzed by the fatty acid synthetase, were not dependent on the presence of CoA. The hydrolysis of acyl- S-multienzyme catalyzed by thioesterase I, the resident chain-terminating component of the fatty acid synthetase, and thioesterase II, a monofunctional mammary gland chain-terminating enzyme, was also independent of CoA availability as was hydrolysis of an acyl- S-pantetheine pentapeptide isolated from the multienzyme. On the basis of these observations we conclude that CoA is required for the elongation of acyl moieties on the fatty acid synthetase but not for their release from the multienzyme.

Coenzyme A requirement for the termination reaction of rat liver fatty acid synthetase.

Highly purified rat liver fatty acid synthetase is completely inhibited when assayed in the presence of a coenzyme A-depleting system such as that catalyzed by phosphotransacetylase, acetyl-CoA synthetase, or ATP citrate lyase. The addition of free CoA causes a reversal of this inhibition. The requirement of free CoA is the same whether acetyl-CoA or butyryl-CoA serves as the primer for fatty acid synthetase. The CoA-depleted and thus inactive fatty acid synthetase system can be reactivated by the addition of a rat brain thioesterase or a rat mammary gland thioesterase II preparation. This reactivation appears to occur in the absence of free CoA. Long chain fatty acids (mainly palmitate) are formed by the thioesterase reactivated system. These results suggest that CoA is required for the termination of the fatty acid synthetase reaction. Possible mechanisms are discussed.

Studies on the mechanism of the effects of experimental inflammation on adaptive synthesis of rat liver fatty acid synthetase

The mechanism of suppression, by experimental inflammation of the usual increase in hepatic fatty acid synthetase activity resulting from fat-free feeding following starvation (adaptive synthesis), was investigated immunochemically. That suppression results from changes in amount of hepatic fatty acid synthetase was shown by the observation that fatty acid synthetase preparations from inflamed and uninflamed animals, exhibiting a wide variety of specific enzyme activities, had identical immunochemical equivalence points. In confirmation of this, the amounts of fatty acid synthetase, determined by radial immunodiffusion in gels containing anti-fatty acid synthetase serum, varied concomitantly with changes in enzyme activity regardless of the relative times of inflammation and onset of adaptive synthesis. Serum insulin levels were not dramatically elevated during the first 48 h of fat-free feeding, but rose markedly thereafter. Inflammation, either alone or combined with fat-free feeding, resulted in increased serum glucose levels, followed by a similar pattern of increased serum insulin levels some 12 h later. Fat-free feeding did not affect serum cortisol levels, but depressed liver cyclic AMP. Inflammation invariably resulted in a marked increase in serum cortisol within 12 h and a concomitant elevation of hepatic cyclic AMP, indicating possible roles for cortisol and cyclic AMP in suppression of hepatic fatty acid synthetase synthesis.

Rat liver fatty acid synthetase: Inactivation of the component enzyme catalysing condensation-CO 2 exchange reaction by chloroacetyl CoA

Summary Rat liver fatty acid synthetase is inhibited by chloroacetyl CoA. The inhibition is rapid and results from the covalent transfer of chloroacetyl group to the enzyme. A stoichiometric relationship exists between the moles of inhibitor bound and the loss of enzyme activity for fatty acid synthesis. Two moles of inhibitor are bound for complete inactivation and this inhibition results from the loss of activity for the critical condensation-CO2 exchange component reaction. These and other data are consistent with the presence of two sites on the enzyme for condensation between enzyme bound acetyl-and malonyl groups.

Coenzyme A is required for rat liver fatty acid synthetase activity.

Inhibition of highly purified rat liver fatty acid synthetase occurs when it is assayed in the presence of the ATP citrate lyase reaction components. Citrate, Mg2+, ATP, and ATP citrate lyase were all necessary for the inhibition to take place. Inhibition was prevented by hydroxycitrate, a competitive inhibitor for ATP citrate lyase. The length of time for the onset of inhibition to take place was proportional to the ratio of ATP citrate lyase activity to the fatty acid synthetase activity. The inhibition was reversed by the addition of coenzyme A. This indicates a reaction mechanism for fatty acid synthetase which involves free coenzyme A. Two possible roles for CoA are discussed, one as an allosteric activator and the other in the cleavage of palmitoyl enzyme in the last step of the reaction.

Physicochemical studies of the rat liver and adipose fatty acid synthetases.

M guanidinium chloride by the analytical ultracentrifuge also indicated that the enzyme consists of two subunits of the same size. The possibility that 6 M guanidinium chloride does not result in complete dissociation of the complex into its ultimate subunits is unlikely since the circular dichroism spectrum of the enzyme in this solvent indicated that the enzyme exists as random coils. Binding studies with [‘4C]acetyl-CoA and [‘4C]malonyl-CoA were consistent with these observations in that they indicated that the 4’-phosphopantetheine and the catalytic centers are associated only with the 220,000 molecular weight subunit. Electron microscopy studies of the rat liver enzyme indicate that the subunit consists of a linear structure 200 A long containing at least four lobes 50 A in diameter. A pseudotetrahedral set of images was also observed and it was proposed that these images resulted from the folding of the linear structures into the pseudotetrahedral form. These observations indicate that the multicatalytic activities of the synthetases and the acyl carrier protein are associated only with the two polypeptide chains. Active enzyme centrifugation studies indicated that the dimer is the only catalytically active form of the enzyme. No larger active form of the enzyme was detected in the presence of the substrates. Furthermore, the monomeric form of the enzyme is inactive. The requirement of the dimer for activity may result from the involvement of two prosthetic groups, or a catalytic center comprising the two polypeptide chains in the catalytic process, or both.

Cell-free translation and partial purification of rat liver fatty acid synthetase mRNA

The cell-free synthesis of rat liver fatty acid synthetase has been demonstrated in a modified reticulocyte lysate translation system. The mRNA was partially purified from membrane-free polysomes by oligo (dT)-cellulose chromatography and subsequent sucrose density gradient centrifugation.

Red blood cell fatty acid synthetase nonidentity with the enzyme from liver

1. 1. Fatty acid synthetase has been purified 680-fold from rat erythrocytes. 2. 2. The enzyme obtained gave one major staining band on disc gel electrophoresis. 3. 3. The molecular weight of the enzyme was estimated to be 310,000 and the enzyme is composed of two half-molecular weight subunits. 4. 4. The red blood cell enzyme synthesizes almost equal amounts of C 14 and C 16 fatty acids (39 and 43%, respectively). 5. 5. The enzyme isolated from rat red blood cells differs from the fatty acid synthetases isolated from the rat adipose tissue, mammary gland, liver and lung in its lack of cross-reactivity with antibody to rat liver fatty acid synthetase. 6. 6. This evidence suggests that the red blood cell fatty acid synthetase is structurally different from that of rat liver.

Identification and quantitation of polysomes synthesizing rat liver fatty acid synthetase during nutritional or hormonal regulation

1. 1. Fatty acid synthetase synthesizing polysomes from rat liver have been identified and quantitated by sucrose density gradient centrifugation with [ 125I]labeled anti-fatty acid synthetase antibody. 2. 2. The [ 125I]labeled antibody binding to nascent fatty acid synthetase peptides demonstrated that the relative content of the fatty acid synthetase mRNA-containing polysomes in rat liver increases dramatically during dietary induction. 3. 3. This increase correlates well with the relative rate of in vivo synthesis of the fatty acid synthetase under the same conditions. 4. 4. The maximum quantity of the synthetase mRNA-containing polysomes was observed at the 12-hr period of dietary induction. Thereafter the quantity of fatty acid synthesizing polysomes declined. 5. 5. Insulin also markedly increased the quantity of synthetase polysomes in diabetic animals, which suggests that this hormone affects regulation of the synthetase at the transcriptional level. 6. 6. Most of the fatty acid synthetase synthesizing polysomes were “free”, rather than membrane-bound.

Identification of sources of hydrogen atoms in fatty acids synthesized using deuterated water and stereospecifically deuterium labelled NADPH by gas chromatographic mass spectrometric analysis

The mechanism of hydrogen incorporation into fatty acids with rat liver fatty acid synthetase was investigated. The distributions of deuterium atoms incorporated into the fatty acids from D2O and stereospecifically deuterium labelled NADPH were determined by mass chromatography. In palmitic acid, 14 deuterium atoms were incorporated from D2O and were found on the even-numbered methylene carbon atoms (two deuterium atoms per carbon atom). Seven of these 14 deuterium atoms were incorporated as the result of deuterium–hydrogen exchange between the methylene group of malonyl-CoA and C2O and D2O. Seven deuterium atoms came from the 4α-position of NADPH and were found on the odd-numbered methylene carbon atoms (one deuterium atom per carbon atom). Seven deuterium atoms from the 4β-position of NADPH were also found on the odd-numbered methylene carbon atoms (one deuterium atom per carbon atom). The stereospecificities of β-ketoacyl reductase and enoyl reductase for NADPH were determined with S-acetoacetyl-N-acetyl-cysteamine and S-crotonyl-N-acetylcysteamine as substrates. The products, S-hydroxybutryl-N-acetyl-cysteamine and S-butyryl-N-acetylcysteamine, respectively, were analysed by gas chromatography mass spectrometry; the results indicated that the β-ketoacyl reductase had B stereospecificity and enoyl reductase had A stereospecificity for NADPH.

Translation and characterization of the fatty acid synthetase messenger RNA.

Fatty acid synthetase messenger RNA was obtained from rat liver polysomal RNA and then injected into Xenopus laevis oocytes. The radioactive fatty acid synthetase protein synthesized in the oocytes was identified by immunoprecipitation with anti-fatty acid synthetase antibody and the immunoprecipitate was then characterized by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel. Co-migration of authentic fatty acid synthetase and the labeled product synthesized in oocytes was observed. Based on sucrose density gradient analysis, the rat liver fatty acid synthetase mRNA has a sedimentation coefficient of approximately 33 S, which agrees with the predicted minimum size necessary to code for the fatty acid synthetase protein. In addition, this mRNA was partially purified with oligo(dT)-cellulose, which indicates that it has a polyadenylate region. The relative in vivo rate of synthesis of fatty acid synthetase and the level of fatty acid synthetase mRNA in liver were also determined during the course of dietary induction of this enzyme. The results indicate that the dietary-induced increase in the level of fatty acid synthetase is probably due to an increased level of the fatty acid synthetase mRNA.

Translation of rat liver fatty acid synthetase mRNA in a cell-free system derived from wheat germ

Total liver polysomes were isolated from rats that had fasted for 48 hr and that then had been re-fed a high-carbohydrate, fat-free diet for 20-24 hr. Indirect immunoprecipitation of the polysomes with purified antibody to rat liver fatty acid synthetase and deproteination on sodium dodecyl sulfate-containing sucrose gradients gave an RNA fraction which, when translated in a cell-free system derived from wheat germ, yielded a major polypeptide of apparent molecular weight 225,000 when the translation products were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The polypeptide was specifically precipitated with antibody against rat liver fatty acid synthetase and competed with unlabeled fatty acid synthetase for binding to the antibody. It was somewhat smaller than native fatty acid synthetase subunits (molecular weight 240,000). The peptide accounted for approximately 65% of the radioactive, antibody-precipitable product, the remainder being peptides in the molecular weight range 100,000-150,000. Synthesis of the polypeptide was optimized with respect to K(+), Mg(2+), and spermine concentrations. The quantity of fatty acid synthetase mRNA obtained by the above procedure and measured by translation was a function of the nutritional state of the animal. The relative activity in fasting rats compared to rats that were re-fed for 12 hr was 1:12. The data suggest that rat liver fatty acid synthetase is synthesized as intact subunits from a large mRNA molecule or molecules.

The effects of experimental inflammation on adaptive synthesis of rat liver fatty acid synthetase

The effects of experimental inflammation, induced by subcutaneous injection of oil of turpentine, on adaptive synthesis of rat liver fatty acid synthetase were investigated. Liver levels of α 1-acid glycoprotein, an “acute-phase” protein known to be synthesized at an accelerated rate as a result of inflammation, were also measured. The increase in fatty acid synthetase activity in livers of rats which were starved and then fed a fat-free diet was suppressed to an extent dependent on the periods between fat-free feeding and inflammation and inflammation and sacrifice. Inflammation induced 2 h after refeeding gave complete suppression, whereas inflammation after 10 h of fat-free feeding had no suppressive effect. When induced 2.5 or 7.5 h after refeeding, inflammation led to partial suppression of the increase in fatty acid synthetase activity. The increase in liver α 1,-acid glycoprotein levels characteristic of inflammation was reduced in animals inflamed 7.5 or 10 h after fat-free feeding, but was unaffected when inflammation was induced 2.5 h after refeeding. The ratio of free to membrane-bound polyribosomes in liver increased from 0.77 in rats which were neither starved nor fed a fat-free diet to 3.31 in rats which were starved and then fed a fat-free diet for 15 h. When inflammation was induced 2.5 h after refeeding, the ratio increased to only 1.74 after 15 h of refeeding. Inflammation resulted in a marked reduction in the level of glycogen in the liver, regardless of the time of induction of inflammation and the dietary status of the animal.

Effect of dietary linoleate on synthesis and degradation of fatty acid synthetase from rat liver.

The induction of fatty acid synthetase activity in rat liver is markedly reduced by feeding a diet containing a source of linoleate. Within 48 h after changing from a fat-free, high carbohydrate diet to one containing 15% by weight of safflower oil, the specific activity of rat liver fatty acid synthetase is approximately 2-fold lower than that from rats fed a fat-free diet througout. In contrast, feeding a diet containing 15% hydrogenated coconut oil, a source of saturated fatty acids, or 5% methyl oleate for the same length of time has little effect on the activity of the enzyme. The rate of synthesis of the enzyme is reduced by safflower oil feeding from the fat-free (control) level and after 48 h is approximately one-half that of the control. The rate of degradation of fatty acid synthetase is markedly increased in the safflower oil-fed animals over the control; the half-lives are 1.8 days and 3.8 days, respectively. It is suggested that polyunsaturated fatty acids, or a product derived from them, may directly or indirectly regulate the transcription or translation (or both) of fatty acid synthetase messenger RNA.