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 C₆-C₁₆; 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.     

Interaction of rat mammary gland thioesterase II with fatty acid synthetase is dependent on the presence of acyl chains on the synthetase

The interaction between rat mammary gland thioesterase II and fatty acid synthetase has been studied by a variety of physicochemical techniques. Pyrene-labeled thioesterase II does not exhibit increased fluorescence anisotropy when mixed with fatty acid synthetase, suggesting that the enzymes do not readily form a complex. Nevertheless, the functional interaction between the enzymes can be easily demonstrated by observing the hydrolysis, by unmodified thioesterase II, of acyl chains from their thioester linkage to the 4-phosphopantetheine of the fatty acid synthetase. This hydrolytic reaction is not inhibited even in the presence of a large excess of fatty acid synthetase with vacant 4'-phosphopantetheine thiols, indicating that interaction occurs only between thioesterase and fatty acid synthetase species which carry acyl chains on the 4'-phosphopantetheine thiols. A novel model system was devised which allowed us to explore the nature of the physical interaction between the two enzymes under conditions where the synthetase was actively engaged in acyl chain assembly. Fatty acid synthetase was treated with phenylmethanesulfonyl fluoride to inhibit its resident thioesterase activity, immobilized via a specific antibody to a column of Sepharose 4B, and exposed to the substrates required for acyl-enzyme assembly. When thioesterase II was introduced to the column, it passed through unretarded even though it efficiently catalyzed hydrolysis of the immobilized S-acyl synthetase en route. These results indicate that the two enzymes associate when an acyl chain is present on the synthetase and that they dissociate rapidly following completion of the catalytic process. Thus, the mammary system differs from that of the avian uropygial gland in which the two enzymes associate to form a stable complex even in the absence of substrates.     

Purification and properties of a thioesterase from lactating rat mammary gland which modifies the product specificity of fatty acid synthetase

An acyl coenzyme A hydrolase (thioesterase II) has been purified to near homogeneity from lactating rat mammary gland. The enzyme is a monomer of molecular weight 33,000 and contains a single active site residue. The enzyme is specific for acyl groups, as acyl-CoA thioesters, containing eight or more carbon atoms and can also hydrolyze oxygen esters. Thioesterase II is capable of shifting the product specificity of rat mammary gland fatty acid synthetase from predominately long chain fatty acids (C14, C16, and C18) to mainly medium chain fatty acids (C8, C10, and C12). Thioesterase II can restore the capacity for fatty acid synthesis to fatty acid synthetase in which the thioesterase component (thioesterase I) has been inactivated with phenylmethanesulfonyl fluoride or removed by trypsinization. No evidence was found of significant levels of thioesterase II in lactating rat liver. The presence of thioesterase II in the lactating mammary gland and the ability of the enzyme to hydrolyze acyl-fatty acid synthetase thioesters of intermediate chain length, are indicative of a major role for this enzyme in the synthesis of the medium chain fatty acids characteristic of milk fat.     

Specific modification of fatty acid synthetase from lactating rat mammary gland by chymotrypsin and trypsin.

Fatty acid synthetase from lactating rat mammary gland after limited proteolysis with chymotrypsin or trypsin synthesizes longer chain fatty acids than those produced by the native enzyme. Of the seven partial reactions of the multienzyme complex, only the thioesterase activity was decreased. The results suggest that modification of the fatty acid synthetase product specificity by chymotrypsin and trypsin results from a specific action of these proteases on the thioesterase component. Trypsin, but not chymotrypsin, cleaved a catalytically active thioesterase from the complex; it thus appears that limited trypsinization will be a useful tool for the isolation of the thioesterase component of the multienzyme.     

Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase

A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.     

Medium chain acyl-thioester hydrolase activity in goat and rabbit mammary gland fatty acid synthetase complexes

The goat mammary gland fatty acid synthetase hydrolysed both medium (C_8:0, C_10:0) and long (C_16:0, C_18:0) chain length acyl CoA esters, whereas the enzyme from rabbit mammary gland only hydrolysed long chain length acyl CoA esters. The medium chain acyl-thioester hydrolase activity of goat mammary gland fatty acid synthetase was much less sensitive to inhibition by phenylmethanesulfonyl-fluorid than the long chain acylthioester hydrolase activity. These results indicate the presence of either two acyl-thioester hydrolases with different specificity or one acyl-thioester hydrolase containing two different active sites.     

Isolation and characterization of a tryptic fragment containing the thioesterase segment of fatty acid synthetase from the uropygial gland of goose.

Trypsin treatment of purified fatty acid synthetase from the uropygial gland of goose released a 33,000 molecular weight peptide from the 270,000 molecular weight synthease. A combination of ammonium sulfate precipitation, Sephadex G-100 gel filtration, anion-exchange chromatography with QAE-Sephadex, and cation-exchange chromatography with cellulose phosphate gave rise to the first homogeneous preparation of the 33,000 molecular weight fragment containing fatty acyl-CoA thioesterase activity. Amino acid composition of this peptide was quite similar to that of the intact fatty acid synthetase except for a lower valine content; a partial specific volume of 0.734 was calculated for the thioesterase fragment. The pH optimum for the thioesterase was near 7.5 and the enzyme showed a high degree of preference for CoA esters of fatty acids with 16 or more carbon atoms. Palmitoyl-CoA inhibited the enzyme and therefore the rate of hydrolysis was not proportional to the amount of protein at low concentrations. Inclusion of bovine serum albumin in the reaction mixture prevented this inhibition. Disregarding the substrate inhibition, an apparent K_m of 5 × 10^?5m and a V of 340 nmol/min/mg were calculated. The thioesterase was inhibited by active serine-directed reagents such as phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate as well as by SH-directed reagents as p-chloromercuribenzoate and N-ethylmaleimide. The isolated thioesterase fragment generated antibodies in rabbits and the antithioesterase inhibited the enzymatic activity of fatty acid synthetase. The antithioesterase showed immunoprecipitant lines with fatty acid synthetase from the uropygial gland and the synthetase from the liver of goose. Anti-fatty acid synthetase prepared against the enzyme from the gland cross-reacted with the thioesterase segment. Even though the synthetase from the uropygial gland synthesizes multimethyl-branched fatty acids in vivo, the thioesterase segment of this synthetase appears to be quite similar to that isolated from the rat.     

Properties of the thioesterase component obtained by limited trypsinization of the fatty acid synthetase multienzyme complex.

The fatty acid synthetase from lactating rat mammary gland is shown to consist of two polyfunctional polypeptides of similar molecular weight (about 220,000); a 4'-phosphopantetheine residue is covalently bound to one, or both subunits. Limited trypsinization of the fatty acid synthetase releases on enzymatically active thioesterase component which has been purified and its properties studied. The thioesterase sediments in the ultracentrifuge as a single component of molecular weight 32,000; its sedimentation coefficient is 2.9 x 10-(13) s its diffusion coefficient 5.0 x 10-(7) cm2 s-(1). The thioesterase also elutes from a column of Sephadex G-75 as a single, symmetrical peak of constant specific activity. However, electrophoresis of the denatured thioesterase in the presence of sodium dodecyl sulfate reveals that the enzyme has been partially nicked during isolation. The kinetic data of the enzyme reaction were studied using palmityl-CoA as a model substrate. Solvent pH was found to affect both Vmax and Km (Km = 0.5 micron at pH 6.6, 2.5 micron at pH 8.0) wereas solvent ionic strength affected Vmax but no Km. The thioesterases from the fatty acid synthetases of rat liver and lactating mammary gland have identical physical properties, identical amino acid compositions, and are immunologically indistinguishable. Both thioesterases hydrolyze long chain, in preference to short chain, thioesters of CoA, an observation consistent with their role in regulation of the chain-terminating step in fatty acid synthesis by the parent multienzyme complexes.     

Amino acid sequence of the serine active-site region of the medium-chain S-acyl fatty acid synthetase thioester hydrolase from rat mammary gland

Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete monomeric enzyme of 29 kDa, regulates the product specificity of the de novo lipogenic systems in certain specialized mammalian and avian tissues, such as mammary and uropygial glands. The amino acid sequence of a 57-residue region containing the active site of the rat mammary gland enzyme has been established by a combination of amino acid and cDNA sequencing. Thioesterase II was radiolabeled with the serine esterase inhibitor [1,3-14C]diisopropyl-fluorophosphate and digested sequentially with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin. A radiolabeled tryptic peptide was isolated and sequenced by automated Edman degradation and the location of the active-site residue established. The amino acid sequence was confirmed by sequencing an overlapping, unlabeled peptide, obtained by V8 digestion of the whole enzyme, and by dideoxynucleotide sequencing of a thioesterase II cDNA clone isolated from a lambda gt11 expression library. The active center contains the motif Gly-Xaa-Ser-Xaa-Gly, characteristic of the serine esterase family of enzymes. A seven-residue region around the essential serine of the rat mammary thioesterase II, Phe-Gly-Met-Ser-Phe-Gly-Ser, is completely homologous with a region of the mallard uropygial thioesterase, recently analyzed by cDNA sequencing, indicating that this is likely to be the active site of the avian enzyme. Overall homology between the mammalian and avian enzymes for the 57-amino-acid residue region is 47% and suggests that the two enzymes may share a common evolutionary origin.     

Inhibition of mammalian fatty acid synthetase activity by NADP involves decreased mobility of the 4'-phosphopantetheine prosthetic group

Mammalian fatty acid synthetase carrying a 3-keto, 3-hydroxy, or 2-enoyl acyl-enzyme intermediate on the 4'-phosphopantetheine thiol is reversibly inhibited by binding of NADP to the enoyl reductase domain. Acyl moieties which can normally leave the enzyme by thioester hydrolysis or by transfer to a CoA acceptor cannot readily be removed from the NADP-inhibited enzyme; in addition, 3-keto or 2-enoyl moieties attached to the enzyme 4'-phosphopantetheine cannot readily be reduced when NADP is replaced by NADPH, even though model substrates can be reduced immediately. Reactivation of the NADP-inhibited 3-ketoacyl-enzyme, by exposure to NADPH, is paralleled by reduction and dehydration of the 3-ketoacyl moiety to a saturated acyl moiety without accumulation of either the 3-hydroxy or 2-enoyl acyl-enzyme intermediates, indicating that once the 4'-phosphopantetheine engages the ketoacyl moiety in the ketoreductase domain, subsequent reactions occur very rapidly. The results are consistent with a hypothesis which proposes that NADP binding to the enoyl reductase domain of fatty acid synthetase carrying an acyl intermediate other than a saturated moiety induces a conformational change in the enzyme that results in decreased mobility of the 4'-phosphopantetheine prosthetic group. Normal mobility of the prosthetic group, essential for transfer of acyl-enzyme intermediates through the active sites of the various functional domains, is restored relatively slowly when NADP is replaced by NADPH. It remains to be determined whether this modulation by pyridine nucleotides observed in vitro plays a role in the regulation of fatty acid synthetase activity in vivo.     

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.     

Subunit structure of the mammalian fatty acid synthetase: further evidence for a homodimer.

Immunochemical procedures and limited proteolysis have been used to investigate the subunit structure of fatty acid synthetase from rat mammary gland. Specific antibodies were raised against the two thioesterase I domains obtained from the fatty acid synthetase by treatment with trypsin. The antibodies precipitated both subunits of the dissociated fatty acid synthetase, indicating that both subunits contained a single thioesterase I domain. An analysis of the time course of limited trypsinization of the fatty acid synthetase, labeled in its two thioesterase I domains with [1,3-¹⁴C] diisopropylphosphofluoridate, indicated that each subunit was susceptible to tryptic attack at identical locations and that the thioesterase I domains occupied a terminal locus at one end of each polyfunctional polypeptide chain. The most plausible explanation for these results is that the mammalian fatty acid synthetase is a homodimer.     

Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis.

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.     

Synthesis of multimethyl-branched fatty acids by avian and mammalian fatty acid synthetase and its regulation by malonyl-CoA decarboxylase in the uropygial gland

Fatty acid synthetase, partially purified by gel filtration with Sepharose 4B from goose liver, showed the same relative rate of incorporation of methylmalonyl-CoA (compared to malonyl-CoA) as that observed with the purified fatty acid synthetase from the uropygial gland. In the presence of acetyl-CoA, methylmalonyl-CoA was incorporated mainly into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8,10-pentamethyl-dodecanoic acid by the enzyme from both sources. Methylmalonyl-CoA was a competitive inhibitor with respect to malonyl-CoA for the enzyme from the gland just as previously observed for fatty acid synthetase from other animals. Furthermore, rabbit antiserum prepared against the gland enzyme cross-reacted with the liver enzyme, and Ouchterlony double-diffusion analyses showed complete fusion of the immunoprecipitant lines. The antiserum inhibited both the synthesis of n-fatty acids and branched fatty acids catalyzed by the synthetase from both liver and the uropygial gland. These results suggest that the synthetases from the two tissues are identical and that branched and n-fatty acids are synthesized by the same enzyme. Immunological examination of the 105,000g supernatant prepared from a variety of organs from the goose showed that only the uropygial gland contained a protein which cross-reacted with the antiserum prepared against malonyl-CoA decarboxylase purified from the gland. Thus, it is concluded that the reason for the synthesis of multimethyl-branched fatty acids by the fatty acid synthetase in the gland is that in this organ the tissue-specific and substrate-specific decarboxylase makes only methylmalonyl-CoA available to the synthetase. Fatty acid synthetase, partially purified from the mammary gland and the liver of rats, also catalyzed incorporation of [methyl-¹⁴C]methylmalonyl-CoA into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid with acetyl-CoA and propionyl-CoA, respectively, as the primers. Evidence is also presented that fatty acids containing straight and branched regions can be generated by the fatty acid synthetase from the rat and goose, from methylmalonyl-CoA in the presence of malonyl-CoA or other precursors of n-fatty acids. These results provide support for the hypothesis that, under the pathological conditions which result in accumulation of methylmalonyl-CoA, abnormal branched acids can be generated by the fatty acid synthetase.     

Xanthomonas campestris RpfB is a fatty Acyl‐CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)‐11‐methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl‐CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl‐CoA ligase, Escherichia coli FadD, in the E. coli ß‐oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3‐hydroxyacyl‐acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl‐ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl‐CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl‐ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.     

Isolation of thioesterase and acyl carrier protein activities liberated by elastase digestion of pigeon liver fatty acid synthetase

Proteolysis of pigeon liver fatty acid synthetase with elastase cleaves the thioesterase component and an acyl carrier protein-containing peptide from the multienzyme complex. These proteins are then separated in one step by gel filtration on a Sephadex G-75 column. Each of the eluted proteins is homogeneous, as determined by polyacrylamide gel electrophoresis. The molecular weight of each has been estimated to be 36,000 and 12,000 daltons, respectively.     

Role of cysteine and 4′-phosphopantetheine in the inactivation of pigeon liver fatty acid synthetase by S-(4-bromo-2,3-dioxobutyl)-coenzyme a

S-(4-bromo-2,3-dioxobutyl)-CoA has been used as an inhibitor of fatty acid synthetase from pigeon liver. This affinity label selectively and irreversibly inhibits the acetyl transacylase and β-ketoacyl synthetase reactions of this multienzyme complex. Binding studies with [³H]-labeled bromodioxobutyl-CoA have established that four mol of the inhibitor are bound per mol of the enzyme complex, and that the radioactivity of this compound is covalently bound to cysteine and 4′-phosphopantetheine moieties. Other partial reactions of fatty acid synthesis are unaffected by bromodioxobutyl-CoA.     

Multiple forms of beta-ketoacyl-acyl carrier protein synthetase in Escherichia coli.

Two forms of beta-ketoacyl-acyl carrier protein (ACP) synthetase (designated I and II) have been identified in extracts of Escherichia coli. Synthetase I corresponds to the condensing enzyme that was studied earlier (GREENSPAN, M.D., ALBERTS, A.W., and VAGELOS, P.R. (1969) J. Biol. Chem. 244, 6477-6485); synthetase II represents a new form of the enzyme. Synthetase II was isolated as a homogeneous protein. It differs from synthetase I in having a higher molecular weight (76,999 versus 66,000), a lower pH optimum (5.5 to 6.1 versus 7.2), and a greater resistance to denaturation by heat. Synthetase II is similar to synthetase I in that both are inactivated by iodoacetamide, and prior incubation of the enzymes with fatty acyl thioesters prevents the inhibitory effect of iodoacetamide. Both also react with a fatty acyl thioester to form an acyl-enzyme intermediate, and the latter reacts with malonyl-ACP to form a beta-ketoacyl thioester. Specificity studies indicated that synthetase II, like synthetase I, has similar affinities with saturated and cis unsaturated fatty acyl thioesters of ACP that are intermediates in the synthesis of saturated and unsaturated fatty acids, respectively. The two synthetases differ only with respect to reactivity with palmitoleyl thioesters: synthetase II has a lower Km and higher Vmax than synthetase I with palmitoleyl-ACP. This finding suggests that synthetase II functions specifically in the elongation of palmitoleyl-ACP to form cis-vaccenyl-ACP. An investigation of synthetases I and II in two classes of unsaturated fatty acid auxotrophs revealed that synthetase I is absent in one class, fabB. Addition of wild type synthetase I to fabB fatty acid synthetase, which synthesizes only saturated fatty acids, permitted this fatty acid synthetase to synthesize unsaturated fatty acids. These experiments indicate that synthetase I plays a critical role in the synthesis of unsaturated fatty acids.     

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [¹⁴C]ATP or [¹⁴C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg²⁺ and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [¹⁴C]ATP or with [³H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C₁₈ fatty acids.     

Biosynthesis of medium-chain fatty acids by mammary epithelial cells from virgin rats.

Epithelial cells were isolated from the undifferentiated mammary glands of mature virgin female rats, and their lipogenic characteristics were studied. These cells synthesized predominantly medium-chain fatty acids, albeit at a low rate. In contrast, whole tissue from mammary glands of virgin rats synthesized predominantly long-chain fatty acids at a relatively higher rate, indicating that the lipogenic activity is dominated by the adipocyte component of the gland. Enzyme assays revealed that thioesterase II, the enzyme which regulates production of medium-chain fatty acids by the fatty acid synthetase, was present at a high activity in the undifferentiated mammary epithelial cells of virgin rats. Immunohistochemical studies confirmed this observation and showed that the regulatory enzyme was present exclusively in the epithelial cells lining the alveolar and ductal elements of the undifferentiated gland. This study demonstrates that the potential to elaborate tissue-specific medium-chain fatty acids is already expressed in the undifferentiated tissue of virgin rats and is not acquired as a result of the differentiation associated with the lactogenic phase of development. In this species mammary epithelial cells apparently synthesize predominantly medium-chain fatty acids at all stages of development, and only the overall rate of synthesis is increased on induction of the fatty acid synthetase during lactogenesis.