Fatty acid synthetase of mammalian brain, liver and adipose tissue regulation by prosthetic group turnover

Jj, Volpe; Pr, Vagelos

doi:10.1016/0005-2760(73)90131-8

Cited by 29 publications

(8 citation statements)

References 20 publications

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“…To verify the values obtained for the activity of the 3-ketoacyl synthase, the partial activity was assayed by two independent methods: (i) the incorporation of (27) has been reported, little is known about how FAS activity is regulated posttranslationally. Since the prosthetic group is indispensable for fatty acid synthesis and its turnover rate is more rapid than that of FAS protein, at least in rat tissues (28)(29)(30), Roncari (3) has proposed that the turnover of 4'-phosphopantetheine is an efficient mode for regulating FAS activity. The loss of 4'-phosphopantetheine from HepG2 FAS suggests that such a mechanism may be operative in the HepG2 cell line.…”

Section: Resultsmentioning

confidence: 99%

Human fatty acid synthase: properties and molecular cloning.

Jayakumar

Tai

Huang

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

204

180

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Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the 13-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and NADPH is a complex process catalyzed by the fatty acid synthase (FAS). In animal tissues, the active synthase is a homodimer of a multifunctional protein that is organized in a head-to-tail fashion, generating two active catalytic centers (1). The seven partial activities and the site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein), are arranged on the multifunctional protein subunit from the amino to carboxyl termini in the following order: f3-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, dehydratase, enoyl reductase, ketoacyl reductase, acyl carrier protein, and thioesterase (1). Most information about the synthase is derived from nonhuman animal studies, so little is known about the human synthase. Investigators who isolated the enzyme from human biopsy tissues (2-5) or cell lines (6) have shown that, except for the enzyme from the SKBR3 cell line (6), all the human FAS preparations had lower activity than the FASs of other animals. This lower activity of the human FAS is comparable to the activities of related human enzymes that are involved in lipogenesis (e.g., pyruvate dehydrogenase, citrate lyase, and glucose-6-phosphate dehydrogenase), which are lower (by a factor of 4-7) in human tissues than in other animal tissues, implying that lipogenesis in humans is highly repressed (7-9).Here we describe the purification and catalytic properties of Assays of FAS and Its Partial Activities. The synthase activity was assayed by measuring the rate of oxidation of NADPH or the incorporation of radiolabeled acetyl-CoA or malonyl-CoA into palmitate (10). The partial activities of FAS were assayed as described earlier: the acetyl/malonyl transacylases (11), dehydratase (12), ,B-ketoacyl synthase (12), f3-ketoacyl reductase (13), ,B-hydroxyacyl enoyl reductase (12), and thioesterase (14). The f3-ketoacyl synthase activity was also determined by measuring the increase in absorbance at 280 nm due to formation of triacetic acid lactone (15).4'-Phosphopantetheine Content. To determine the 4'-phosphopantetheine content of FAS, we developed a simple and sensitive method based on spectrophotometric measurement of the phenylthiocarbamoyl (PTC) derivative of taurine released after performic acid oxidation and hydrolysis of the protein. PTC-taurine can be separated from other PTC-amino acids by using reverse-phase high-performance liquid chromatography (HPLC) and following the absorbance at 254 nm. In this system,...

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Section: Resultsmentioning

confidence: 99%

Human fatty acid synthase: properties and molecular cloning.

Jayakumar

Tai

Huang

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

204

180

View full text Add to dashboard Cite

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“…In both instances the completed F AS polypeptide chains are modified by posttranscriptional events. Thus, a rapid turnover of the 4'-phosphopante theine prosthetic group of the fatty acid synthetase (holo�apoenzyme) from rat liver, brain, and adipose tissue has been observed (163). During starvation the rate of this prosthetic group exchange diminishes in liver but not in brain (163).…”

Section: Adaptive Control Mechanismsmentioning

confidence: 99%

Control Mechanisms in the Synthesis of Saturated Fatty Acids

Bloch¹,

Vance²

1977

Annu. Rev. Biochem.

319

140

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“…Workers in several laboratories have reported the isolation from animal FAS of peptides containing 4'-phosphopantetheine ranging in molecular weight from 6,000 to 100,000 (9,10,17,29 therefore appears that mammalian FAS is composed of only two peptides of essentially identical molecular weight. The formation of lower molecular weight peptides probably occurs by proteolysis during the procedure utilized to dissociate the FAS.…”

Section: Resultsmentioning

confidence: 99%

“…The changes in enzyme activity noted during starvation and diabetes are due to changes in the amount of FAS and not to activation or inactivation of existing enzyme (11,(12)(13)(14)(15)(16). The amount of FAS present in both liver and adipose tissue is largely regulated by the rate of FAS synthesis in these organs (11,14,17 (22). Briefly, livers from rats starved for 48 hr and then refed a fat-free diet were homogenized in 3 volumes of 0.2 M potassium phosphate buffer, pH 7.0, containing 1 mM dithiothreitol and 1 mM EDTA.…”

mentioning

confidence: 99%

Regulation of synthesis of hepatic fatty acid synthetase: binding of fatty acid synthetase antibodies to polysomes.

Alberts¹,

Strauss²,

Hennessy³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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Mammalian fatty acid synthetase was shown to be composed of two peptides, molecular weight 240,000, after dissociation with sodium dodecyl sulfate. Rat liver polysomes that synthesize fatty acid synthetase were identified by sucrose gradient analysis of polysomes that had been reacted with 12 I-labeled antibody against fatty acid synthetase. The binding of 125I-labeled antibody to polysomes was found to correlate with the rate of hepatic fatty acid synthesis in various nutritional conditions. Fatty acid synthetase (FAS) has been identified in all biological systems that catalyze the de novo synthesis of long chain fatty acids from acetyl CoA and malonyl CoA (1-3). Although the mechanisms of the sequential reactions that constitute the FAS are similar in various biological systems, the structures of FAS differ dramatically in prokaryotes and eukaryotes (1). Thus, the Escherichia colt FAS is composed of at least seven peptides which represent the individual enzyme components and the acyl carrier protein (1, 2, 4). The yeast and animal FAS (molecular weight 2.2 X 106 and 4.8 X 105, respectively), on the other hand, readily dissociate into only two high-molecular-weight peptide components (5-7). Harsh dissociation conditions have been reported to produce lower molecular weight peptides from the yeast (8) and animal FAS (9, 10).Another distinguishing feature of the animal system is the response of FAS to nutritional and hormonal perturbations.Thus, FAS activity is depressed in starved animals; upon refeeding a fat-free diet the enzyme activity increases to super high levels (1,(11)(12)(13)(14). Low levels of FAS activity are also found in diabetic animals, and this condition is reversed by the administration of insulin (15,16). The changes in enzyme activity noted during starvation and diabetes are due to changes in the amount of FAS and not to activation or inactivation of existing enzyme (11,(12)(13)(14)(15)(16). The amount of FAS present in both liver and adipose tissue is largely regulated by the rate of FAS synthesis in these organs (11,14,17 (22). Briefly, livers from rats starved for 48 hr and then refed a fat-free diet were homogenized in 3 volumes of 0.2 M potassium phosphate buffer, pH 7.0, containing 1 mM dithiothreitol and 1 mM EDTA. The 100,000 X g supernatant from the homogenate was subjected to ammonium sulfate precipitation at 35% saturation. The resulting precipitate was dissolved in the homogenizing buffer and diluted with the buffer to a final protein concentration of 10 mg/ ml. To this was added 6 volumes of alumina Cy in 1 mM dithiothreitol at a concentration of 5 mg/ml. After stirring 5 min at room temperature, the suspension was centrifuged and the pellet discarded. The supernatant was adjusted with solid ammonium sulfate to 45% saturation and the resulting precipitate was dissolved in a minimal volume of homogenizing buffer. This was then applied to a sepharose-6 B column equilibrated with 0.25 M sodium phosphate, pH 7.0, containing 1 mM dithiothreitol and 1 mM EDTA. The fractions from...

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Fatty acid synthetase of mammalian brain, liver and adipose tissue regulation by prosthetic group turnover

Cited by 29 publications

References 20 publications

Human fatty acid synthase: properties and molecular cloning.

Human fatty acid synthase: properties and molecular cloning.

Control Mechanisms in the Synthesis of Saturated Fatty Acids

Regulation of synthesis of hepatic fatty acid synthetase: binding of fatty acid synthetase antibodies to polysomes.

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