Uropygial Gland Of Goose Research Articles

Cloning and expression of rat pancreatic β-cell malonyl-CoA decarboxylase

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

Peroxisome proliferator-activated receptor γ1 (PPAR-γ1) as a major PPAR in a tissue in which estrogen induces peroxisome proliferation

Estradiol administration induces peroxisome proliferation and the production of 3-hydroxy fatty acid pheromones in the uropygial glands of the duck, but not in the goose gland, which does not produce such pheromones. We isolated a peroxisome proliferator-activated receptor (PPAR)γ1 cDNA from a duck uropygial gland cDNA library. Northern blots revealed two transcripts, PPARγ1 and γ2, and showed that PPARγ was expressed at higher levels than PPARα in the uropygial gland of the duck. Although PPARγ2 was expressed in both duck and goose uropygial gland, PPARγ1 was expressed only in the duck gland, which responds to estrogen by peroxisome proliferation. In NIH 3T3 transfected cells, PPARγ1 was activated by peroxisome proliferators such as Wy-14 643, clofibric acid and Ly-171 883 causing induction of the target marker gene. By cotransfection with a plasmid containing α- cis-retinoic acid receptor RXRα, the induction increased up to 9-fold. These results suggest that PPARγ1 may be involved in peroxisome proliferation while PPARγ2 may be involved in lipid metabolism.

Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.

Molecular Cloning, Nucleotide Sequence, and Tissue Distribution of Malonyl-CoA Decarboxylase

Malonyl-CoA decarboxylase was purified from goose uropygial gland, reduced, carboxymethylated, and digested with trypsin. Several peptides were purified by high performance liquid chromatography and their amino acid sequences determined. Oligonucleotide probes were prepared based on their amino acid sequences. Size-selected RNA from the goose uropygial gland was used to construct cDNA libraries in lambda gt11 and pUC9 vectors. Immunological screening of the lambda gt11 cDNA library yielded one clone, lambda DC1, which contained a 2.2-kilobase pair insert; hybridization with the synthetic oligonucleotide probes confirmed its identity as malonyl decarboxylase. Screening of the pUC9 cDNA library with the insert of lambda DC1 as a probe detected one clone, pDC2, with an insert of 2.9 kilobase pairs. The nucleotide sequences of the two cDNAs revealed an open reading frame encoding a polypeptide of 462 amino acids. The deduced amino acid sequence was confirmed as malonyl-CoA decarboxylase by matching it to the amino acid sequences of three tryptic peptides derived from mature enzyme. Northern blot analysis of mRNA from goose brain, kidney, liver, lung, and gland revealed malonyl-decarboxylase mRNA of 3000 nucleotides. Since clone pDC2 contains a 2928-nucleotide insert, it represents nearly the full length of mRNA. Brain, kidney, lung, and liver contained less than 1% of the malonyl-CoA decarboxylase mRNA in the gland. Southern blot analysis of genomic DNA showed a single band in both liver and gland, suggesting that malonyl-CoA decarboxylase is a single copy gene.

Specific modification of the condensation domain of fatty acid synthase and the determination of the primary structure of the modified active site peptides

Fatty acid synthase from the uropygial gland of goose was inactivated by iodoacetamide with a second-order rate constant of 1.3 m −1 s −1 at pH 6.0 and 25 °C. Of the seven component activities of the synthase, only the condensation activity was significantly inhibited by iodoacetamide modification. Since preincubation of the enzyme with acetyl-CoA, but not with malonyl-CoA, protected the enzyme from inactivation by iodoacetamide, it is suggested that iodoacetamide probably modified the primer-binding thiol group at the condensation active site. Determination of the stoichiometry of modification was done using [1- 14C]iodoacetamide that was purified by high-performance liquid chromatography. Graphical analysis of the data showed that binding of 1.2 carboxamidomethyl groups per subunit of fatty acid synthase would result in complete inhibition of the enzyme activity, suggesting that there is one condensation domain per subunit of fatty acid synthase. Analysis of the tryptic peptide map of the enzyme that was modified with [1- 14C]iodoacetamide in the presence and absence of acetyl-CoA revealed that acetyl-CoA prevented the labeling of a major radioactive peptide and a minor radioactive peptide. These two peptides were purified by high-performance liquid chromatography. Amino acid analysis of these two peptides revealed that the major radioactive peptide contained S-carboxymethylcysteine while the minor radioactive peptide did not. However, the latter peptide contained β-alanine, suggesting that this peptide was from the acyl carrier protein segment of fatty acid synthase and that the iodoacetamide treatment resulted in modification of the pantetheine thiol, although to a lower extent than the primer-binding thiol. The sequence of the primer-binding active site peptide from the condensation domain was H 2NGlyProSerLeuSerIleAspThrAlaCys(carboxamidomethyl) XSerSerLeuMetAlaLeuGluAsnAlaTyrLysCOOH, the first reported sequence of the condensation active site from a vertebrate fatty acid synthase. The acyl carrier protein segment showed extensive sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the phosphopantetheine attachment, and the sequence was H 2NAspValSerSerLeuAsnAlaAspSerThrLeuAla AspLeuGlyLeuAspSer(4′-phosphopante-theine)LeuMetGlyVal GluValArgCOOH.

Sequence of a tryptic peptide from the NADPH binding site of the enoyl reductase domain of fatty acid synthase

Fatty acid synthase from the uropygial gland of goose was inhibited by treatment with pyridoxal 5′-phosphate by selectively modifying a lysine residue at the NADPH binding site of the enoyl reductase domain ( A. J. Poulose and P. E. Kolattukudy (1980) Arch. Biochem. Biophys. 201, 313–321). Distribution of radioactivity in tryptic peptides generated from the synthase treated with pyridoxal 5′-phosphate/NaB 3H 4 in the presence and absence of 2′-monophosphoadenosine-5′-diphosphoribose, which protects the enzyme from inactivation by pyridoxal phosphate, showed that modification of one specific peptide was prevented by the protector. This peptide was purified by a combination of Sephadex G-25 column chromatography, anion-exchange chromatography, and high-performance liquid chromatography. The primary structure of this peptide is ValPheThrThrValGlySerAlaGluLys(Pxy)Arg.

Molecular cloning of cDNA sequences for avian malic enzyme. Nutritional and hormonal regulation of malic enzyme mRNA levels in avian liver cells in vivo and in culture.

A double-stranded cDNA library constructed from the total poly(A+) RNA of goose uropygial gland was screened for recombinants containing sequences complementary to malic enzyme mRNA. Replicate arrays of 1400 colonies were hybridized independently with 32P-labeled cDNAs copied from two populations of hepatic RNA derived from tissues which differed by about 35-fold with respect to the relative synthesis of malic enzyme. Forty-eight of the colonies which gave differential signals were further screened by hybrid-selected translation. DNA from one of these contained an insert of 970 base pairs and selected an mRNA which directed the synthesis of malic enzyme in a cell-free system. The malic enzyme sequences were subcloned into the single-stranded bacteriophage M13mp8. The subclones were used to prepare 32P-labeled single-stranded hybridization probe. Northern analysis indicated that malic enzyme mRNA from both goose and chicken is about 2100 bases in length. Hepatic malic enzyme mRNA concentration is stimulated 30- to 50-fold or more when neonatal chicks or goslings, respectively, are fed for 24 h. When added to chick embryo hepatocytes in culture, triiodothyronine stimulated malic enzyme mRNA accumulation by more than 100-fold. Glucagon inhibited the thyroid hormone-stimulated accumulation of malic enzyme mRNA by 99%. In all instances, malic enzyme mRNA concentration was closely correlated with the relative rate of malic enzyme synthesis. These results suggest that nutritional and hormonal regulation of malic enzyme synthesis occurs at the pretranslational level.

Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration.

A double-stranded cDNA library was constructed using total poly(A)+ RNA from the goose uropygial gland. Clones containing sequences complementary to fatty acid synthase mRNA were initially identified by colony hybridization with a 32P-labeled cDNA transcribed from RNA enriched for fatty acid synthase mRNA. Identity of the fatty acid synthase clones was confirmed by hybrid-selected translation. Mature fatty acid synthase mRNA is approximately 16 kilobases in length. When unfed neonatal goslings were fed for 24 hr, relative synthesis of hepatic fatty acid synthase increased more than 42-fold. Concomitantly, hepatic fatty acid synthase mRNA levels increased 70-fold. Thus, nutritional regulation of the synthesis of hepatic fatty acid synthase probably occurs at the pretranslational level. The availability of a specific probe for fatty acid synthase mRNA should allow us to analyze the regulation of expression of this gene during development, by nutrition and by hormones in both liver and uropygial gland.

Purification and characterization of Acyl-CoA carboxylase from the goose uropygial gland which produces multimethyl-branched acids and evidence for its identity with avian Acetyl-CoA carboxylase

Acyl-CoA carboxylase was purified from the 140,000 g supernatant of the goose uropygial gland extract by means of Sepharose 4B-CL gel filtration, ammonium sulfate precipitation, and affinity chromatography with monomeric avidin-Sepharose 4B-CL. The purified enzyme showed a pH optimum of 8 and had a specific activity ranging from 2–8 μmol/min/mg protein for acetyl-CoA. Sodium dodecyl sulfate-electrophoresis showed a single band corresponding to a molecular weight of 238,000. Carboxylase activity was stimulated threefold by 20 m m citrate. Maximal activity was observed with 25 m m bicarbonate, 10 m m Mg 2+, 3 m m ATP, and 1 to 2 m m acyl-CoA. The enzyme carboxylated acetyl-CoA, propionyl-CoA, butyryl-CoA, pentanoyl-CoA, and hexanoyl-CoA, with a V of 8.8, 5.7, 0.9, 0.04, and 0.03 μmol/min/mg, respectively; K m values for the five CoA esters were quite similar. The carboxylated products from these substrates were analyzed by high-performance liquid chromatography. This carboxylase was inhibited by sodium and chloride ions. Chemical modification of the enzyme with pyridoxal-5′-phosphate showed inhibition of activity that was time and concentration dependent. The inhibition was reversed by dilution except when treated with sodium borohydride before dilution. Acetyl-CoA partially (40%) protected the enzyme from inhibition, whereas 3′-dephosphoacetyl-CoA, which showed a K m 3.5 times that of acetyl-CoA, was much less efficient in protecting the enzyme against inactivation by pyridoxal phosphate. These results suggest that the ϵ-amino group of a lysine residue is involved in binding acetyl-CoA via interaction with the 3′-phosphate. Chemical modification of the enzyme with phenylglyoxal showed inhibition of activity that was time and concentration dependent. However, none of the substrates protected the enzyme from inactivation; citrate partially protected the enzyme, possibly by changing the configuration of the enzyme. Amino acid analysis of the protein showed striking similarities with carboxylases purified from other animals. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the gland enzyme showed fusion of precipitation lines with the enzymes from goose liver and chicken liver. These results strongly support the conclusion that the uropygial gland, which synthesizes multimethyl-branched acids, employs the same carboxylase as that present in other tissues.

Specific acetylation of essential lysine residues in malonyl-CoA decarboxylase

1. 1. Malonyl-CoA decarboxylase from the uropygial gland of goose was inactivated by treatment with low concentrations of acetic anhydride in Tris-acetate buffer. 2. 2. The degree of inactivation depended on the concentration of the reagent and time of incubation. 3. 3. Treatment of the enzyme with [1- 14C]acetic anhydride resulted in covalent attachment of the label exclusively to the decarboxylase protomer. 4. 4. Evidence is presented that the label incorporated into the protein was exclusively in N-ϵ-acetyllysine. Graphical analysis suggested that acetylation of about two lysine residues/subunit would be required for complete inactivation of the enzyme. 5. 5. Malonyl-CoA. acetyl-CoA, propionyl-CoA and methylmalonyl-CoA afforded partial protection to the enzyme against inactivation by acetic anhydride. 6. 6. 3'-Dephosphomalonyl-CoA was found to be an alternate substrate for the enzyme with approximately the same K m and V values as for malonyl-CoA. 7. 7. This alternate substrate also partially protected the enzyme from inactivation, whereas adenine nucleotides with 2'-, 3'-, or 5'-phosphates did not protect the enzyme. 8. 8. These results suggest that a lysine residue acetylated by acetic anhydride is essential for the enzyme activity and that it is probably at or near the active site. The results are consistent with the suggestion that the enzyme is composed of four identical subunits.

Evidence that the coenzyme a requirement for avian fatty acid synthase is not for the ermination reaction

1. 1. Fatty acid synthase from goose uropygial gland was inhibited when CoA was scavenged with ATP citrate lyase. 2. 2. This inhibition was reversed by the addition of CoA, 3'-dephospho-CoA, 1, N 6-etheno-CoA and pantetheine but not by desulfo-CoA, pantethine or mercaptoethanol suggesting that the structural features of pantetheine including the free thiol group are essential for the reversal. 3. 3. The S-acyi fatty acid synthase thioester hydrolase from the uropygial glands of mallards, which hydrolytically removes acyi chains from fatty acid synthase of goose uropygial gland in the absence of CoA, did not reverse this inhibition, suggesting that the CoA depletion does not inhibit termination reaction. 4. 4. However, triacetic acid lactone synthesis by the fatty acid synthase was inhibited by the scavenging of CoA, raising the possibility that the condensation and/or the transacylase reactions may require CoA.

Primary structure of a chymotryptic peptide containing the “active serine” of the thioesterase domain of fatty acid synthase

“Active serine” of the thioesterase domain of fatty acid synthase from the goose uropygial gland was selectively labeled with [1,3- 3H]diisopropylfluorophosphate and the chymotryptic peptide containing this active serine was purified to homogeneity by a combination of gel filtration, cation exchange chromatography and high performance liquid chromatography. The primary structure of this active site peptide, Ser-Phe-Gly-Ala-Cys-Val-Ala-Phe, is remarkably homologous to the “active serine” containing peptide of human plasmin.

Production and utilization of butyryl-CoA by fatty acid synthetase from mammalian tissues

Butyrate constitutes 25 to 40% of the fatty acids formed by fatty acid synthetase purified from cow mammary gland, rat mammary gland, rat liver, and rat adipose tissue. In contrast, the extent of formation of this acid by the enzyme complex from chicken liver, pigeon liver, goose uropygial gland, and yeast is only 2 to 5%. Most of the butyrate produced is esterified to CoA, while most of the longer-chain acids formed are as free acids. The formation of butyrate appears to be a physiological process as judged from its formation in high amounts in rat liver slices. Butyrate formation is correlated with the ability of the enzyme from the different sources to utilize butyryl-CoA as primer. It is argued that butyryl- S-Enzyme formed as an intermediate during the course of fatty acid synthesis either undergoes chain elongation by condensation with malonyl-CoA or has the butyryl group transferred to CoA by a transacylation reaction. The concentration of malonyl-CoA or free CoA then determines which of the two competing processes is favored.

13] Fatty acid synthase from the uropygial gland of goose

13] Fatty acid synthase from the uropygial gland of goose

Goose fatty acid synthetase mRNA.

The fatty acid synthetase of animal tissues consists of two identical subunits (Mr = 250,000), each of which is a multienzyme protein containing domains for the acyl carrier peptide and the seven different catalytic activities required for the conversion of acetyl-CoA and malonyl-CoA to palmitate. Total poly(A+) RNA was isolated from goose uropygial gland and translated in a cell-free rabbit reticulocyte system. The translation products contained a polypeptide having the same molecular weight as the native synthetase subunits; this protein was specifically recognized by anti-synthetase antibodies and could be competed by excess native synthetase for antibody binding. Fractionation of the poly(A+) RNA by sucrose gradient centrifugation showed that synthetase mRNA is very large, repeatedly exhibiting a sedimentation coefficient of 35 S. Gel electrophoresis of this purified mRNA fraction following glyoxylation showed the presence of several species of RNA, one of which correlated well with the in vitro translation of the synthetase. This mRNA species has a molecular weight of 2.95 X 10(6), which is large enough to code for a protein with a molecular weight of 250,000. These results confirm the multifunctional nature of the synthetase and indicate that the synthetase subunit must arise as a single polypeptide chain synthesized from one contiguous mRNA.

Chemical modification of an essential lysine at the active site of enoyl-CoA reductase in fatty acid synthetase

Fatty acid synthetase from goose uropygial gland was inactivated by treatment with pyridoxal 5′-phosphate. Malonyl-CoA and acetyl-CoA did not protect the enzyme whereas NADPH provided about 70% protection against this inactivation. 2′-Monophospho-ADP-ribose was nearly as effective as NADPH while 2′-AMP, 5′-AMP, ADP-ribose, and NADH were ineffective suggesting that pyridoxal 5′-phosphate modified a group that interacts with the 5′-pyrophosphoryl group of NADPH and that the 2′-phosphate is necessary for the binding of the coenzyme to the enzyme. Of the seven component activities catalyzed by fatty acid synthetase only the enoyl-CoA reductase activity was inhibited. Inactivation of both the overall activity and enoyl-CoA reductase of fatty acid synthetase by this compound was reversed by dialysis or dilution but not after reduction with NaBH 4. The modified protein showed a characteristic Schiff base absorption (maximum at 425 nm) that disappeared on reduction with NaBH 4 resulting in a new absorption spectrum with a maximum at 325 nm. After reduction the protein showed a fluorescence spectrum with a maximum at 394 nm. Reduction of pyridoxal phosphate-treated protein with NaB 3H 4 resulted in incorporation of 3H into the protein and paper chromatography of the acid hydrolysate of the modified protein showed only one fluorescent spot which was labeled and ninhydrin positive and had an R f identical to that of authentic N 6-pyridoxyllysine. When [4- 3H]pyridoxal phosphate was used all of the 3H, incorporated into the protein, was found in pyridoxyllysine. All of these results strongly suggest that pyridoxal phosphate inhibited fatty acid synthetase by forming a Schiff base with the ϵ-amino group of lysine in the enoyl-CoA reductase domain of the enzyme. The number of lysine residues modified was estimated with [4- 3H]pyridoxal-5′-phosphate/NaBH 4 and by pyridoxal-5′-phosphate/NaB 3H 4. Scatchard analysis showed that modification of two lysine residues per subunit resulted in complete inactivation of the overall activity and enoyl-CoA reductase of fatty acid synthetase. NADPH prevented the inactivation of the enzyme by protecting one of these two lysine residues from modification. The present results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.

Presence of one essential arginine that specifically binds the 2′-phosphate of NADPH on each of the ketoacyl reductase and enoyl reductase active sites of fatty acid synthetase

Two arginine modifying reagents, phenylglyoxal and 2,3-butanedione, inactivated fatty acid synthetase from goose uropygial gland. This inactivation could be partially prevented by NADP, 2′-AMP, and 2′,5′-ADP, whereas acetyl-CoA and/or malonyl-CoA provided very little protection. Ketoacyl reductase and enoyl reductase activities of fatty acid synthetase showed similar inactivation by phenylglyoxal and butanedione and protection by only NADP and its 2′-phosphate-containing analogs. Furthermore, 2′-AMP was found to be a competitive inhibitor of overall fatty acid synthetase, ketoacyl reductase, and enoyl reductase with apparent K i values of 1.4, 0.2, and 14 m m, respectively. These results suggest that binding of NADPH to fatty acid synthetase involves specific interaction of the 2′-phosphate with the guanidino group of arginine residues at the active site of the two reductases. Quantitation of the number of arginine residues modified revealed that 4 out of 106 arginine residues per subunit of the synthetase showed high reactivity toward phenylglyoxal. Scatchard analysis showed that two rapidly reacting arginine residues had no effect on the catalytic activity, while modification of two additional arginine residues resulted in complete loss of enzyme activity. Under these conditions, of the seven partial reactions of fatty acid synthetase, only the ketoacyl reductase and enoyl reductase activities were inhibited by phenylglyoxal. The differential reversal of inhibition of the two reductases and the overall activity of fatty acid synthetase, resulting from dialysis of the modified enzyme, suggested that both ketoacyl reductase sites and enoyl reductase sites are required for the full expression of fatty acid synthetase activity. The results of the present chemical modification studies are consistent with the hypothesis that each subunit of fatty acid synthetase contains one ketoacyl reductase and one enoyl reductase and suggest that one essential arginine is present at each of these active sites.

Malonyl-CoA decarboxylase from the mammary gland of lactating rat: Purification, properties and subcellular localization

Malonyl-CoA decarboxylase (EC 4.1.1.9) was purified 500–600-fold from the mammary gland extracts by (NH 4) 2SO 4 precipitation, gel filtration with Sepharose 4B, anion-exchange chromatography with QAE-Sephadex, and chromatography with NADP-Agarose. This enzyme (spec. act. 200–300 nmol/ min per mg protein) had a molecular weight of approx. 170 000. It did not cross-react with rabbit antiserum prepared against either fatty acid synthetase from the mammary gland or malonyl-CoA decarboxylase from the uropygial gland of goose. The decarboxylase showed a pH optimum near 8.5–9.0 and a K m of 0.33 mM, decarboxylated neither malonic acid nor methylmalonyl-CoA and was inhibited by thiol directed reagents but not by avidin. Sucrose density gradient centrifugation of the gland homogenate showed that the major peak of decarboxylase activity coincided with that of cytochrome oxidase. Breakage of mitochondria released > 80% of the decarboxylase activity into the 105 000 × g supernatant, suggesting that malonyl-CoA decarboxylase may be located in the mitochondrial matrix.

Evidence for an “active serine” in each fatty acid synthetase peptide

Ultracentrifugally homogeneous fatty acid synthetase was isolated from the uropygial gland of goose by a one-step purification procedure. Formation of fatty acids from malonyl-CoA and hydrolysis of palmitoyl-CoA catalyzed by the synthetase were inhibited to an equal extent by diisopropylfluorophosphate. With labeled inhibitor, it was shown that one mole of the inhibitor was covalently attached per mole of the subunit of the enzyme. Sodium dodecyl sulphate electrophoresis showed that all of the label was contained in a 270,000 M.Wt peptide. That the active serine was not at the loading site was suggested by the observations that neither acetylation nor malonylation of the enzyme affected the reaction of the enzyme with the inhibitor and acetylation or malonylation of the enzyme was not affected by this inhibitor. Thus, each fatty acid synthetase peptide is shown to have one active serine which most probably is at the chain terminating active site of the peptide.