Rat Liver Fatty Acid Synthetase Research Articles

Subunits of fatty acid synthetase complexes: Comparative study of enzyme activities and properties of the half-molecular weight nonidentical subunits of fatty acid synthetase complexes obtained from rat, human, and chicken liver and yeast

Rat, human, and chicken liver and yeast fatty acid synthetase complexes were dissociated into half-molecular weight nonidentical subunits of molecular weight 225,000–250,000 under the same conditions as used previously for the pigeon liver fatty acid synthetase complex [Lornitzo, F. A., Qureshi, A. A., and Porter, J. W. (1975) J. Biol. Chem. 250, 4520–4529]. The separation of the half-molecular weight nonidentical subunits I and II of each fatty acid synthetase was then achieved by affinity chromatography on Sepharose ϵ-aminocaproyl pantetheine. The separations required, as with the pigeon liver fatty acid synthetase, a careful control of temperature, ionic strength, pH, and column flow rate for success, along with the freezing of the enzyme at −20 °C prior to the dissociation of the complex and the loading of the subunits onto the column. The separated subunit I (reductase) from each fatty acid synthetase contained β-ketoacyl and crotonyl thioester reductases. Subunit II (transacylase) contained acetyl- and malonyl-coenzyme A: pantetheine transacylases. Each subunit of each complex also contained activities for the partial reactions, β-hydroxyacyl thioester dehydrase (crotonase), and palmitoyl-CoA deacylase. The specific activities of a given partial reaction did not vary in most cases more than twofold from one fatty acid synthetase species to another. The rat and human liver fatty acid synthetases required a much higher ionic strength for stability of their complexes and for the reconstitution of their overall synthetase activity from subunits I and II than did the pigeon liver enzyme. On reconstitution by dialysis in high ionic strength potassium phosphate buffer of subunits I and II of each complex, 65–85% of the control fatty acid synthetase activity was recovered. The rat and human liver fatty acid synthetases cross-reacted on immunoprecipitation with antisera. Similarly, chicken and pigeon liver fatty acid synthetases crossreacted with their antisera. There was, however, no cross-reaction between the mammalian and avian liver fatty acid synthetases and the yeast fatty acid synthetase did not cross-react with any of the liver fatty acid synthetase antisera.

Isolation, purification, and properties of mammalian and avian liver and yeast fatty acid synthetase acyl carrier proteins

Acyl carrier proteins were isolated from rat, human, pigeon, and chicken liver and yeast fatty acid synthetase complexes. These proteins were separated from the other proteins of subunit I of each complex by ultrafiltration after dialysis of subunit I for 3 h against low ionic strength buffer [Qureshi et al. (1974) Biochem. Biophys. Res. Commun. 60, 158–165]. Subunit I of each fatty acid synthetase was previously separated from subunit II by affinity chromatography on Sepharose ϵ-aminocaproyl pantetheine and subsequent sucrose density gradient centrifugation. The separated acyl carrier proteins were then subjected to gel filtration on a Sephadex G-50 column. The proteins obtained from each fatty acid synthetase were homogeneous with respect to size and charge on gel filtration, paper and disc gel electrophoresis, and chromatography on diethylaminoethyl-cellulose. The physical properties and the ability to accept acetyl and malonyl groups from acetyl- and malonyl-CoA in the presence of transacylase were similar to those of Escherichia coli acyl carrier protein. These proteins ranged in molecular weight from 7500 to 10,000. Each of the acyl carrier proteins showed the presence of β-alanine and each yielded acetyl- and malonyl-A 1 and A 2 peptic peptides, thus indicating the presence of a 4′-phosphopantetheine prosthetic group in each. They differed somewhat from each other in amino acid composition, but each had a high number of negatively charged (aspartate and glutamate) amino acid residues.

On the 4'-phosphopantetheine content of chicken and rat liver fatty acid synthetases.

The finding that animal synthetases are complexes consisting of two polypeptide chains (Stoops, J.K., Arslanian, M.J., Oh, Y.H., Vanaman, T.C., and Wakil, S.J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1940-1944) led us to investigate their 4'-phosphopantetheine content. We have found that the chicken and rat synthetases contain 1.6 to 2.2 mol of 4'-phosphopantetheine per mol of the complex. The implications of this finding concerning the structure of the complex and the biosynthetic pathway of fatty acid synthesis are discussed.

Studies on the synthesis of rat liver fatty acid synthetase.

The synthesis of the multienzyme complex rat liver fatty acid synthetase was investigated utilizing modifications of methods developed in the laboratory of Schimke (Schimke, R. T. (1964) J. Biol. Chem. 239, 3808-3817 and Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). The relative amounts of radioactivity from a pulse of labeled lysine appearing in polypeptides derived from purified synthetase complex can be measured compensating for the varying amounts of lysine per polypeptide chain. The results show that labeled amino acid is incorporated into polypeptides derived from the complex at heterogeneous rates. However, 10 to 15 hours after the administration of a pulse, the amount of label per lysine residue in these polypeptides is identical. The results support the previously proposed model of this multienzyme complex (Tweto, J., Dehlinger, P., and Larrabee, A. R. (1972) Biochem. Biophys. Res. Commun. 48, 1371-1377). The previous work and that reported here suggests the existence of a pool of synthetase subunits which is an obligatory intermediate in both synthesis and turnover of the complex. The results obtained in this work are consistent with this model if the exchange of subunits into the intact complex is a relatively slow process requiring several hours to reach equilibrium.

Effects of thyroid hormones on enzymes involved in fatty acid and glycerolipid synthesis

The influence of thyroid hormones on lipid biosynthesis was studied after administration of L-thyroxine to rats for 5 days. Their weights remained the same as those of control animals, despite an approximately 3-fold increment in plasma L-thyroxine and L-triiodothyronine concentrations. The activity of acetyl-CoA carboxylase and fatty acid synthetase as well as incorporation of tritium into fatty acids were depressed significantly in epididymal adipose tissue and enhanced significantly in livers of thyroxine-treated rats. Using antibodies specific against rat liver fatty acid synthetase, it was determined that the changes in activity of this multienzymic complex were due to alterations in amount of enzyme protein. In the presence of optimal concentrations of fatty acids, radioactive sn-glycero-3-phosphate, and co-substrates, total glycerolipid synthesis (defined in this study as the sum of newly formed radioactive mono- and diacyl-sn-glycero-3-phosphate, diglyceride, and triglyceride) was decreased significantly in adipose tissue and increased in liver and heart. Thus, administration of thyroid hormone results in tissue-specific alterations in lipid biosynthesis which, at least in the case of fatty acid synthetase, are due to changes in enzyme protein content.

Adaptive synthesis of rat liver fatty acid synthetase: Evidence for [formula omitted] formation of active enzyme from inactive protein precursors and 4′-phosphopantetheine

Evidence is presented in support of the hypothesis that an important step in the adaptive synthesis of fatty acid synthetase is the conversion of inactive enzyme precursors to active enzyme via the incorporation of the 4′-phosphopantetheine prosthetic group. Fatty acid synthetase activity was generated in vitro when CoA or E. coli acyl carrier protein was incubated with enzymatically inactive extracts from livers of rats fed a fat-free diet for 0–5 hr following starvation, and a factor present in liver extracts from rats refed for more than 6 hr. When ( 14C)-CoA, labelled in the pantetheine moiety, was used in the above system, radioactivity was incorporated into a protein bound form, from which it could be released by mild alkaline hydrolysis.

Coordinate control of rat liver lipogenic enzymes by insulin

Recent evidence has established that insulin is required for the dietary induction of rat liver fatty acid synthetase [ Proc. Nat. Acad. Sci. USA 69, 3516 (1972)]. Since other hepatic lipogenic enzymes as well as fatty acid synthetase exhibit coordinate adaptation to nutritional changes [ Advan. Enzyme Regul. 10, 187(1972)], the role of insulin in the dietary induction of these enzymes has been investigated. When a high-carbohydrate, fat-free diet was fed to diabetic rats previously fasted for 48 hr, insulin was shown to be required for the dietary induction of acetyl-CoA carboxylase, citrate cleavage enzyme, malic enzyme, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fatty acid synthetase, and glucokinase. Activity of serine dehydrase, selected as a model gluconeogenic enzyme, was increased in diabetic rats, whereas insulin treatment reduced the activity of this enzyme during the course of refeeding. The behavior of serine dehydrase was consistent with its gluconeogenic role. The activity of the cytosol isocitrate dehydrogenase did not change during refeeding in the diabetic or insulin-treated diabetic rat. Glucagon, the physiological antagonist of insulin, inhibited the increase in activity of each of the lipogenic enzymes requiring insulin for induction. Our results indicate that insulin is required for the coordinate regulation of the lipogenic enzymes of mammalian liver.

Effect of vitamin B-12 deprivation on the rates of synthesis and degradation of rat liver fatty acid synthetase

To characterize the basis for the increased hepatic fatty acid synthetase activity in vitamin B-12 deprivation, the content and rates of synthesis and degradation for the enzyme were determined. Animals were in a dietary steady state on normal chow or a B-12-deprived diet; animals on the latter diet were further divided into a “supplemented” group given B-12 and those “B-12-deprived.” The B-12-deprived animals had tissue B-12 depletion. Both total and specific activity of fatty acid synthetase were increased in the B-12-deprived animals, and this was due to increased enzyme protein. Rates of synthesis and degradation were studied in each group after a pulse of 20 μCi of l-[U- 14C]leucine. Radioactivity was determined in the immunoprecipitate of the purified enzyme. Relative rates of synthesis in the B-12-deprived group were increased 8.8-fold over the normal and 3.6-fold over the B-12-supplemented group. Degradation of hepatic fatty acid synthetase was 63 hr ( t 1 2 ) in the normal and 65 hr in the B-12-supplemented group. The t 1 2 in the B-12-deprived group was 35 hr. Degradation rates for the soluble protein pool were not affected by B-12 deprivation. The rate constant for synthesis of hepatic fatty acid synthetase in the B-12-deprived group was 14-fold that of the normal and 6-fold that of the B-12-supplemented animals. Thus, although vitamin B-12 deprivation results in increased rate of degradation of fatty acid synthetase, enzyme synthesis is markedly increased yielding a severalfold net increase in synthetase content and activity.

Studies on the adaptive synthesis of rat liver fatty acid synthetase

The adaptive synthesis of fatty acid synthetase in the livers of rats fed a fat-free diet following 48 hr of fasting has been studied using immunochemical methods. The development of fatty acid synthetase activity during adaptive synthesis occurs about 3 hr following feeding, whereas the synthesis of material precipitable by anti-fatty acid synthetase serum, as judged by the incorporation of 3H-labeled amino acids into the immunoprecipitate, commenced within 1 hr. Extracts of liver of rats fed a fat-free diet for 1–3 hr following fasting contain increasing amounts of material which competes with purified fatty acid synthetase for antibody binding sites, even though they have no fatty acid synthetase activity. This suggests the presence of enzymatically inactive precursors of fatty acid synthetase in the liver extracts. The incorporation of [ 14C]pantothenate into fatty acid synthetase during adaptive synthesis follows the same pattern as the development of enzyme activity, indicating that these enzymatically inactive precursors of fatty acid synthetase may represent an apoenzyme which is converted to the enzymatically active holoenzyme by the incorporation of the 4′-phosphopantetheine prosthetic group. The subcellular site of synthesis of fatty acid synthetase was shown to be in the pool of polysomes that are not membrane bound, rather than in the rough endoplasmic reticulum.

Fatty acid synthetase complex: Selective inactivation by phenylmethylsulphonyl fluoride

Reaction of pigeon and rat liver fatty acid synthetases with phenylmethylsulphonyl fluoride at pH 7.0 results in rapid and complete loss of activity for fatty acid synthesis. Acetyl and malonyl transacylation, two reductions, dehydration and condensation-CO 2 exchange reactions are not appreciably altered in the modified enzyme. However, the deacylation of palmityl CoA is completely inhibited. Complete inactivation results in the incorporation of about 1.9 moles of 14C-phenylmethylsulphonyl groups/mole of the enzyme complex. These results suggest that either two moles of a fatty acyl deacylase or two deacylases with different fatty acyl chain length specificities may be functional in the enzyme complex.

Effect of dietary change on the rates of synthesis and degradation of rat liver fatty acid synthetase

Investigations were carried out to determine whether diet-induced changes in the level of rat liver fatty acid synthetase are due to changes in the rate of synthesis or degradation of this enzyme. Assays for radioactivity in fatty acid synthetase were carried out through isolation of the enzyme by a standard purification procedure or by immunoprecipitation with antiserum prepared specifically against rat liver fatty acid synthetase. Studies on the incorporation of radioactive leucine into the fatty acid synthetase during steady-state conditions showed that the relative rate of synthesis of this enzyme was decreased to one-fifth the normal value in the fasted state and increased to 14.3 times the normal value in rats refed a fat-free diet. Studies on the degradation of the fatty acid synthetase, reported as half-life of the enzyme, yielded identical values, 69 hr, for normal animals, as well as for animals refed a fat-free diet. The rates of synthesis of enzyme ( k s ) calculated from these half-life values and the enzyme content present in the liver in a given nutritional state agreed well with the relative rates of synthesis. On the basis of these results it is concluded that changes in the level of liver fatty acid synthetase which are diet induced are due primarily to changes in the rate of synthesis rather than degradation of this enzyme. However, transient changes in the rate of degradation may also be important during the transition period, particularly in the case of fasting.

Acyl Carrier Protein: XIX. AMINO ACID SEQUENCE OF LIVER FATTY ACID SYNTHETASE PEPTIDES CONTAINING 4′-PHOSPHOPANTETHEINE

Abstract Overlapping tryptic, peptic, thermolytic, and Pronase peptides containing 4'-phosphopantetheine were isolated from rat liver fatty acid synthetase. Characterization of these peptides and a 4'-phosphopantetheine dipeptide obtained after dilute acid hydrolysis permitted the elucidation of the amino acid sequence of 13 residues. Five out of the 13 residues are identical with those in the corresponding region of Escherichia coli acyl carrier protein.

Relative turnover rates of subunits of rat liver fatty acid synthetase

The subunits of the multienzyme complex rat liver fatty acid synthetase are degraded with half lives which are related to their molecular weight in a manner similar to that found in the laboratory of Schimke for soluble, membrane and ribosomal proteins from rat liver. The rates of turnover of all the subunits of fatty acid synthetase appear to be slower than the rate of exchange of the enzyme prosthetic group, 4′-phosphopantetheine.

The Effect of Fasting on Synthesis and 4′-Phosphopantetheine Exchange in Rat Liver Fatty Acid Synthetase

Abstract The level of rat liver fatty acid synthetase is greatly decreased upon fasting. This report presents several additional observations of the effect of fasting on that enzyme. (a) The rate of synthesis is reduced during 48 hours of fasting and is 20% of the steady state rate after 12 hours. (b) The apparent half-life of the enzyme is decreased from about 70 hours to about 20 hours. (c) The exchange of the prosthetic group, 4'-phosphopantetheine, is largely eliminated, perhaps by effects on the mechanism of attachment or hydrolysis. (d) Feeding a protein-free diet mimics the effect of starvation on the enzyme levels.

Kinetics of binding of reduced nicotinamide adenine dinucleotide phosphate to the rat liver fatty acid synthetase complex

A 5-fold variation in each of the dissociation constants for the binding reactions between NADPH and the two pairs of NADPH-binding sites possessed by rat liver fatty acid synthetase occurred in response to changes in phosphate buffer concentration over the range 0.12–0.20 m. Estimates of the rate constants for the binding reaction and for the dissociation of the enzyme-coenzyme complex have been obtained for each pair of binding sites over a range of buffer concentrations. This was accomplished using stopped-flow fluorescence enhancement. The rate constants for the binding reactions were found to be approximately 1 × 10 7 m −1, sec −1 and 1.2 × 10 6 m −1, sec −1 for the reactions occurring at the stronger and weaker pairs of binding sites, respectively. Furthermore, these values remained substantially constant over the range of phosphate buffer concentrations tested. Conversely, the rate constants for the dissociation reactions of the enzyme-NADPH complex at the stronger and weaker pairs of binding sites were found to vary in response to buffer concentration changes over the ranges 1–9 sec −1 and 10–30 sec −1, respectively.

Relative turnover rates of proteins and peptides of rat liver fatty acid synthetase

The double label method of Arias et al . (J. Biol. Chem., 244 , 3303, 1969) has been adapted to measure the relative turnover rates of proteins and peptides derived from rat liver fatty acid synthetase from starved and refed animals. Differential rates of turnover were not detected in either proteins from polyacrylamide gel electrophoresis, nor in peptides obtained by tryptic digestion.

Protein Turnover and 4'-Phosphopantetheine Exchange in Rat Liver Fatty Acid Synthetase

Abstract The rate of turnover of rat liver fatty acid synthetase has been measured in animals tested under steady state conditions. The value obtained for the half-life varied from 71 to 108 hours. The exchange rate of the covalently bound prosthetic group, 4'-phosphopantotheine, with unbound pantothenate compounds was found to be more than an order of magnitude greater than the half-life of the enzyme complex. A model is proposed in which the prosthetic group, or some small portion of the enzyme complex containing the prosthetic group, is periodically removed from and replaced on the larger fatty acid synthetase moiety, such action taking place many times before an individual complex is catabolized. Moreover, this exchange is not part of the catalytic function of an enzyme complex, but may be a means of control of over all fatty acid synthetase activity.

Isolement de la synthetase des acides gras a partir de microsomes de foies de rats

Some rat liver fatty acid synthetase has been isolated from microsomal fraction. Enzyme activity and electro micrography of purified enzyme show that the microsomal enzyme was the same as that of the supernatant. These results are discussed in relation to the role of microsomes in the de novo synthesis of fatty acids synthesis in the cell.

Mechanism of Binding of Reduced Nicotinamide Adenine Dinucleotide Phosphate to Vertebrate Fatty Acid Synthetases: SITES AND TYPE OF BONDING, CONFORMATION OF COENZYME, AND BINDING OF ANALOGUES

The techniques of fluorescence enhancement and fluorescence polarization were used to study the binding of NADPH and structurally similar pyridine nucleotides to pigeon and rat liver fatty acid synthetases. NADPH, on binding to the enzyme, changes from a closed to an open conformation and the nicotinamide ring becomes associated with hydrophobic groups of the protein. The orientation of NADPH when bound to enzyme is such as to permit the transfer of radiant energy from tryptophan residues of the protein to the nicotinamide moiety of NADPH. The binding affinity of the enzyme for the coenzyme is considerably reduced by removal of the 2'-ribose phosphate (NADH), and by oxidation of the pyridine ring (NADP), but it is only slightly decreased by alteration of substituents on the adenine and pyridine rings (deamino-NADP and acetylpyridine-NADP). Enzyme-bound NADH is not aligned with the enzyme in the same manner as NADPH. The binding of NADPH to enzyme is competitively inhibited by pyridine nucleotide analogues, and at higher concentrations, by the ions of inorganic salts structurally unrelated to NADPH. Hence, electrostatic forces of attraction between charged groups on the coenzyme and the enzyme appear to have an important role in maintaining enzyme-coenzyme association.

Stereospecificity of the Transfer of Hydrogen from Reduced Nicotinamide Adenine Dinucleotide Phosphate to the Acyl Chain in the Dehydrogenase-catalyzed Reactions of Fatty Acid Synthesis

Abstract Tritium from NADP3H was incorporated into the fatty acids (mainly palmitic acid) synthesized from acetyl and malonyl coenzyme A by purified pigeon and rat liver fatty acid synthetases. The quantity of tritium incorporated into the product was nearly the same from the A and B forms of NADP3H. These compounds were prepared from tritiated isocitric and glutamic acids, respectively. The quantity of radioactivity incorporated into fatty acids per mole of NADPH oxidized was slightly less than half the quantity of radioactivity present per mole of isocitric or glutamic acid. On the assumption that the two types of reduction in fatty acid synthesis (β-ketoacyl-enzyme and α,β-unsaturated acyl-enzyme) proceed stereospecifically, as do all other pyridine nucleotide dehydrogenase reactions, these results indicate that one reduction proceeds by the transfer of hydrogen from the A side of position 4 of the pyridine ring of NADPH, and the other reduction proceeds by transfer from the B side. This deduction was confirmed by the performance of partial reactions with model substrates. When acetoacetyl coenzyme A or S-acetoacetyl-N-acetylcysteamine was reduced to the β-hydroxybutyryl ester with fatty acid synthetase and tritiated NADPH, tritium was incorporated into the product only from the B form of the coenzyme. When S-crotonyl-N-acetylcysteamine was similarly reduced to the butyryl ester, tritium was incorporated into product only from the A form of NADPH. Thus, the reduction of β-ketoacyl-enzyme in fatty acid synthesis results in the transfer of hydrogen from the B side of NADPH, and the reduction of α,β-unsaturated acyl-enzyme results in the transfer of hydrogen from the A side of NADPH.