Characterization and partial purification of indole-3-butyric acid synthetase from maize (Zea mays)

In previous work it has been shown that the route from indoleacetic acid (IAA) to indolebutyric acid (IBA) is likely to be a two-step process with an unknown intermediate designated ‘product X′. Our objective was to characterize and purify enzyme activities that are involved in these reactions. Indole-3-butyric acid synthetase was isolated and characterized from light-grown maize seedlings (Zea mays L.), which were able to synthesize IBA from indole-3-acetic acid (IAA) with ATP and acetyl-CoA as cofactors. The enzyme activity is most likely located on the membranes of the endoplasmic reticulum, as shown by means of aqueous two-phase partitioning and sucrose density gradient centrifugation, with subsequent marker enzyme analysis. It was possible to solubilize the enzyme from the membranes with a detergent (CHAPS) and high concentrations of NaCl. The molecular mass of solubilized IBA synthetase was ca 31 kDa and its isoelectric point was at pH 4.8. The enzyme forming the reaction intermediate had a molecular mass of only 20 kDa and it seemed to be located on different membranes. Inhibition experiments with reducing agents and sulfhydryl reagents indicated that no sulfhydryl groups or disulfide bridges were present in the active centre of IBA synthetase. KCN inhibited the enzyme activity completely, and sodium azide by about 50%. Substrate analogs. such as 1-IAA, 2,4-dichlorophenoxyacetic acid, phenylacetic acid, and naphthaleneacetic acid, inhibited IBA formation to a high extent. Experiments with tunicamycin gave evidence that the enzyme is not a glycoprotein. These findings were confirmed by affinity chromatography with Concanavalin A. where the enzyme did not bind to the matrix. Further purification of the IBA synthetase on an ATP-affinity column resulted in a more than 1 000-fold purification compared to the microsomal membranes. IBA synthetase activity was also present in other plant families. Our results present further evidence that IBA is synthesized by a two-step mechanism involving two different enzyme activities.     

Regulation of IBA synthetase from maize (Zea mays L.) by drought stress and ABA

The rate of indole-3-butyric acid (IBA) synthesis in maize seedlingsis dependent on the culture conditions of the plants. When theseedlings were grown on filter paper soaked with different amountsof water, the activity of IBA synthetase differed strongly.High amounts of water (150 and 200 ml per bowl) inhibited IBAsynthesis completely in vitro, whereas 30 and 50 ml water perbowl increased the activity dramatically. Under conditions whereIBA synthetase was inhibited (150 ml H₂O), an increase of enzymeactivity was observed when abscisic acid (ABA) was exogenouslyadded in concentrations between 510^–4 to 510^–7M. Under ‘drought’ conditions (50 ml H₂O per bowl)the same ABA concentrations were inhibitory. Jasmonic acid andsalicylic acid also enhanced IBA synthetase activity to someextent, whereas indole-3-acetic acid (IAA) and kinetin had noeffect. Activity could also be enhanced by osmotic stress (NaCIand sorbitol), but not under temperature stress. In accompanyinginvestigations the endogenous contents of IAA, IBA, and ABAunder the different culture conditions have been determinedas well as the energy charge of the seedlings. Similar observationshave been made with Amaranthus, wheat and pea seedlings Key words: Abscisic acid, Amaranthus paniculatus, drought stress, inole-3-butyric acid biosynthesis, Pisum sativum, Triticum aestivum, Zea mays     

Indole-3-butyric acid in plant growth and development

Within the last ten years it has been established by GC-MS thatindole-3-butyric acid (IBA) is an endogenous compound in a variety ofplant species. When applied exogenously, IBA has a variety of differenteffects on plant growth and development, but the compound is stillmainly used for the induction of adventitious roots. Using moleculartechniques, several genes have been isolated that are induced duringadventitious root formation by IBA. The biosynthesis of IBA in maize(Zea mays L.) involves IAA as the direct precursor. Microsomalmembranes from maize are able to convert IAA to IBA using ATP andacetyl-CoA as cofactors. The enzyme catalyzing this reaction wascharacterized from maize seedlings and partially purified. The invitro biosynthesis of IBA seems to be regulated by several externaland internal factors: i) Microsomal membranes from light-grownmaize seedlings directly synthesize IBA, whereas microsomal membranesfrom dark-grown maize plants release an as yet unknown reaction product,which is converted to IBA in a second step. ii) Drought and osmoticstress increase the biosynthesis of IBA maybe via the increaseof endogenous ABA, because application of ABA also results in elevatedlevels of IBA. iii) IBA synthesis is specifically increased byherbicides of the sethoxydim group. iv) IBA and IBA synthesizingactivity are enhanced during the colonization of maize roots with themycorrhizal fungus Glomus intraradices. The role of IBA forcertain developmental processes in plants is discussed and somearguments presented that IBA is per se an auxin and does notact via the conversion to IAA.     

Indole-3-butyric acid synthesis in ecotypes and mutants of Arabidopsis thaliana under different growth conditions

Although IBA is a naturally occurring auxin, its role in plant development is still under debate. In this study a set of Arabidopsis mutants was used to analyze the biosynthesis of IBA in vitro. The mutants chosen for this study can be classified as: (1) involvement in auxin metabolism, transport or synthesis (amt1, aux1, ilr1, nit1, rib1, sur1, trp1-100); (2) other hormones possibly involved in the regulation of IBA synthesis (aba1, aba3, eto2, fae1, hls1, jar1); (3) photomorphogenesis (det1, det2, det3); and (4) root architecture (cob1, cob2, scr1). In addition, two transgenic lines overexpressing the IAA glucose synthase (iaglu) gene from maize were analyzed. The ecotypes No-0 and Wassilewskija showed the highest IBA synthetase activity under control conditions, followed by Columbia, Enkheim and Landsberg erecta. In the mutant lines IBA synthetase activity differed in most cases from the wild type, however no particular pattern of up- or down-regulation, which could be correlated to their possible function, was found. For rib1 mutant seedlings it was tested whether reduced IBA synthetase activity correlates with the endogenous IBA levels. Free IBA differed only depending on the culture conditions, but gave no clear correlation with IBA synthetase activity compared to the wild type. Since drought and osmotic stress as well as abscisic acid (ABA) application enhanced IBA synthesis in maize, it was tested whether IBA synthetase from Arabidopsis is also inducible by drought stress conditions. This was confirmed for the two ecotypes Col and Ler which showed different IBA synthetase activity when cultivated with various degrees of drought stress. IBA synthetase was also determined in photomorphogenic mutants under different light regimes. Induction of IBA synthetase in det1 and det3 plants was found under short day plus a red light pulse or in the dark, respectively. The results are discussed with respect to the functions of the mutated genes.     

Malonyl-CoA synthetase, encoded by ACYL ACTIVATING ENZYME13, is essential for growth and development of Arabidopsis

Malonyl-CoA is the precursor for fatty acid synthesis and elongation. It is also one of the building blocks for the biosynthesis of some phytoalexins, flavonoids, and many malonylated compounds. In plants as well as in animals, malonyl-CoA is almost exclusively derived from acetyl-CoA by acetyl-CoA carboxylase (EC 6.4.1.2). However, previous studies have suggested that malonyl-CoA may also be made directly from malonic acid by malonyl-CoA synthetase (EC 6.2.1.14). Here, we report the cloning of a eukaryotic malonyl-CoA synthetase gene, Acyl Activating Enzyme13 (AAE13; At3g16170), from Arabidopsis thaliana. Recombinant AAE13 protein showed high activity against malonic acid (K(m) = 529.4 ± 98.5 μM; V(m) = 24.0 ± 2.7 μmol/mg/min) but little or no activity against other dicarboxylic or fatty acids tested. Exogenous malonic acid was toxic to Arabidopsis seedlings and caused accumulation of malonic and succinic acids in the seedlings. aae13 null mutants also grew poorly and accumulated malonic and succinic acids. These defects were complemented by an AAE13 transgene or by a bacterial malonyl-CoA synthetase gene under control of the AAE13 promoter. Our results demonstrate that the malonyl-CoA synthetase encoded by AAE13 is essential for healthy growth and development, probably because it is required for the detoxification of malonate.     

Long-term regulation by theophylline of fatty acid synthetase, acetyl-CoA carboxylase and lipid synthesis in cultured glial cells.

The long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase and of fatty acid and sterol synthesis was studied in C-6 glial cells in culture. When theophylline (10(-3) M) was added to the culture medium of these cells, rates of lipid synthesis from acetate and activities of synthetase and carboxylase became distinctly lower than in cells that were untreated. This effect appeared after approximately 12 h, and after 48 h enzymatic activities were reduced approx. 2-fold and rates of lipid synthesis from acetate 3- to 4-fold. The likelihood that the decrease in fatty acid synthesis from acetate was caused by the decrease in activities of fatty acid synthetase and acetyl-CoA carboxylase was established by several observations. These indicated that the locus of the effect probably did not reside at the level of acetate uptake into the cell, alterations in acetate pool sizes or conversion of acetate to acetyl-CoA. Moreover, de novo fatty acid synthesis was found to be the predominant pathway in these glial cells, whether treated with theophylline or not. The mechanism of the effect of theophylline on fatty acid synthetase was shown by immunochemical techniques to involve an alteration in content of enzyme rather than in catalytic efficiency. The change in content of fatty acid synthetase was shown by isotopic-immunochemical experiments to involve a decrease in synthesis of the enzyme. The mechanism whereby theophylline leads to a decrease in lipogenesis and in the synthesis of fatty acid synthetase may not be mediated entirely by inhibition of phosphodiesterase and an increase in cyclic AMP levels, because dibutyryl cyclic AMP (10(-3) M) only partially reproduced the effect.     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

Mechanisms by which tumor necrosis factor stimulates hepatic fatty acid synthesis in vivo

We have previously shown that bolus intravenous administration of tumor necrosis factor (TNF) to normal rats results in a rapid (within 90 min) stimulation of hepatic fatty acid synthesis, which is sustained for 17 hr. We now demonstrate that TNF stimulates fatty acid synthesis by several mechanisms. Fatty acid synthetase and acetyl-CoA carboxylase (measured after maximal stimulation by citrate) were not higher in livers from animals that had been treated with TNF 90 min before study compared to controls. In contrast, 16 hr after treatment with TNF, fatty acid synthetase was slightly elevated (35%) while acetyl-CoA carboxylase was increased by 58%. To explain the early rise in the hepatic synthesis of fatty acids, we examined the regulation of acetyl-CoA carboxylase. The acute increase in fatty acid synthesis was not due to activation of acetyl-CoA carboxylase by change in its phosphorylation state (as calculated by the ratio of activity in the absence and presence of 2 mM citrate). However, hepatic levels of citrate, an allosteric activator of acetyl-CoA carboxylase, were significantly elevated (51%) within 90 min of TNF treatment. TNF also induces an acute increase (within 90 min) in the plasma levels of free fatty acids. However, hepatic levels of fatty acyl-CoA, which can inhibit acetyl-CoA carboxylase, did not rise 90 min following TNF treatment and were 35% lower than in control livers by 16 hr after TNF. These data suggest that TNF acutely regulates hepatic fatty acid synthesis in vivo by raising hepatic levels of citrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and in Vivo Biosynthesis of Indole-3-Butyric Acid in Corn (Zea mays L.)

Indole-3-butyric acid (IBA) was identified as an endogenous compound in leaves and roots of maize (Zea mays L.) var Inrakorn by thin layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. Its presence was also confirmed in the variety Hazera 224. Indole-3-acetic acid (IAA) was metabolized to IBA in vivo by seedlings of the two maize varieties. The reaction product was identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry after incubating the corn seedlings with [¹⁴C]IAA and [¹³C₆]IAA. The in vivo conversion of IAA to IBA and the characteristics of IBA formation in two different maize varieties of Zea mays L. (Hazera 224 and Inrakorn) were investigated. IBA-forming activity was examined in the roots, leaves, and coleoptiles of both maize varieties. Whereas in the variety Hazera 224, IBA was formed mostly in the leaves, in the variety Inrakorn, IBA synthesis was detected in the roots as well as in the leaves. A time course study of IBA formation showed that maximum activity was reached in Inrakorn after 1 hour and in Hazera after 2 hours. The pH optimum for the uptake of IAA was 6.0, and that for IBA formation was 7.0. The K_m value for IBA formation was 17 micromolar for Inrakorn and 25 micromolar for Hazera 224. The results are discussed with respect to the possible functions of IBA in the plant.     

Substrate inhibition of pigeon liver fatty acid synthetase and optimum assay conditions for over-all synthetase activity

The effects of the substrates acetyl-CoA, malonyl-CoA, and NADPH on the activity of pigeon liver fatty acid synthetase have been studied over a wide range of concentrations. Double-reciprocal coordinate plots for each of the substrates have been found to be linear at low concentrations. At higher concentrations two of the substrates, acetyl-CoA and malonyl-CoA, inhibit the rate of fatty acid synthesis. This double substrate inhibition is apparently of a competitive type. Inhibition by acetyl-CoA is very strong as compared to that by malonyl-CoA. At a 4:1 ratio of acetyl- to malonyl-CoA, inhibition is about 75%, whereas at a 4:1 ratio of malonyl- to acetyl-CoA fatty acid synthesis proceeds at the maximum rate.These results are consistent with the hypothesis that a competition between acetyl-CoA and malonyl-CoA occurs for the occupany of the 4′- phosphopantetheine site, a prosthetic group of the synthetase complex, and possibly also for the hydroxyl binding site (or sites). The relative concentrations of these substrates and the binding constants for each then determine whether these sites are occupied by acetyl or malonyl groups, and whether inhibition of fatty acid synthesis occurs. Based on our results, assays for pigeon liver fatty acid synthetase activity should be conducted at substrate concentrations of 15 μm, 60 μm, and 100 μm for acetyl-CoA, malonyl-CoA, and NADPH, respectively.     

Effects of Manganese Deficiency and Added Cerium on Nitrogen Metabolism of Maize

Manganese is one of the essential microelements for plant growth, and cerium is a beneficial element for plant growth. However, whether manganese deficiency affects nitrogen metabolism of plants and cerium improves the nitrogen metabolism of plants by exposure to manganese-deficient media are still unclear. The main aim of the study was to determine the effects of manganese deficiency in nitrogen metabolism and the roles of cerium in the improvement of manganese-deficient effects in maize seedlings. Maize seedlings were cultivated in manganese present Meider's nutrient solution. They were subjected to manganese deficiency and to cerium chloride administered in the manganese-present and manganese-deficient media. Maize seedlings grown in the various media were measured for key enzyme activities involved in nitrogen metabolism, such as nitrate reductase, glutamate dehydrogenase, glutamine synthetase, and glutamic-oxaloace transaminase. We found that manganese deficiency restricted uptake and transport of NO(3)(-), inhibited activities of nitrogen-metabolism-related enzymes, such as nitrate reductase, glutamine synthetase, and glutamic-oxaloace transaminase, thus decreasing the synthesis of chlorophyll and soluble protein, and inhibited the growth of maize seedlings. Manganese deficiency promoted the activity of glutamate dehydrogenase and reduced the toxicity of excess ammonia to the plant, while added cerium relieved the damage to nitrogen metabolism caused by manganese deficiency in maize seedlings. However, cerium addition exerted positively to relieve the damage of nitrogen metabolism process in maize seedlings caused by exposure to manganese-deficient media.     

Acetyl-coenzyme A carboxylase in maize leaves

Purified chloroplasts from mesophyll and bundle sheath cells of maize leaves have been shown to be the location of acetyl-CoA carboxylase. In disrupted chloroplasts the enzyme was recovered in the stromal fraction, along with protein-bound biotin; acetyl-CoA carboxylase activity did not require a membrane component. Mg²⁺ and ATP are required for activity and sulfhydryl protecting agents enhance stability of the enzyme. Acetyl-CoA carboxylase activity was independent of leaf development in cell-free extracts of maize. Comparison of acetyl-CoA carboxylase activity with [¹⁴C]acetate incorporation into lipids, in isolated chloroplasts from developing leaves of maize, indicate that acetyl-CoA carboxylase is not limiting fatty acid synthesis.     

Regulation of palmitic acid synthesis in cultured glial cells: effects of glucocorticoid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and sterol synthesis.

Abstract— C-6 glial cells in culture were utilized to define the role of glucocorticoid in the regulation of palmitic acid synthesis and the important lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase. Particular emphasis was given to fatty acid synthetase which exhibited more than a 50% reduction in specific activity when cells were exposed to hydrocortisone (10 μg/ml) for 1 week. Coordinate changes in acetyl-CoA carboxylase activity and in palmitic acid (and sterol) synthesis from acetate accompanied the alterations in fatty acid synthetase. Immunochemical techniques were utilized to show that the decrease in synthetase activity involved an alteration in enzyme content, not in catalytic efficiency. The changes in content of fatty acid synthetase were caused by alterations in enzyme synthesis. Glucocorticoids may regulate fatty acid synthetase in C-6 glial cells by a mechanism similar to that suggested for adipose tissue. The inhibition of palmitic acid synthesis may be relevant to other effects of glucocorticoids on developing brain.     

Synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase

Pigeon liver fatty acid synthetase has been found to catalyze the formation of palmitic acid from malonyl-CoA and NADPH in the absence of acetyl-CoA. Radio-chemical and spectral assays show that the activity of the complex in the absence of acetyl-CoA is about 25–30% of the activity in the presence of this compound. Initial velocities were determined for a series of reactions in which the malonyl-CoA concentration was varied over a range of 5–200 μm at a fixed NADPH concentration of 100μm and vice versa. No inhibitory effects of one substrate over the other were found. However, when the synthesis of fatty acids was studied in the presence of acetyl-CoA, a significant inhibitory effect of malonyl-CoA was observed. It has also been shown that the fatty acid synthetase synthesizes triacetic lactone from malonyl-CoA in the absence of NADPH and acetyl-CoA. No evidence was obtained for the direct decarboxylation of malonyl-CoA to acetyl-CoA in this reaction. Hence it is proposed that decarboxylation of the malonyl moiety bound covalently to 4′-phosphopantetheine occurs to yield acetyl-4′-phosphopantetheine. Further, it is proposed that the acetyl moiety of the latter compound is transferred to the cysteine site of the enzyme complex and that fatty acid synthesis proceeds in the presence of NADPH as proposed by Phillips et al. [Arch. Biochem. Biophys.138, 380 (1970)]. In the absence of NADPH triacetic lactone is formed.     

Auxins in the development of an arbuscular mycorrhizal symbiosis in maize

While the levels of free auxins in maize (Zea mays L.) roots during arbuscular mycorrhiza formation have been previously described in detail, conjugates of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with amino acids and sugars were neglected. In this study, we have therefore determined free, ester and amide bound auxins in roots of maize inoculated with Glomus intraradices during early stages of the colonization process. Ester conjugates of IAA and IBA were found only in low amounts and they did not increase in AM colonized roots. The Levels of IAA and IBA amide conjugates increased 20 and 30 days past inoculation (dpi). The formation of free and conjugated IBA but not IAA was systemically induced during AM colonization in leaves of maize plants. This implicated a role for auxin conjugate synthesis and hydrolysis during AM. We have therefore investigated the in vivo metabolism of 3H-labeled IBA by TLC but only slight differences between control and AM-inoculated roots were observed. The activity of auxin conjugate hydrolase activity measured with three different putative substrates showed a decrease in infected roots compared to controls. The fluorinated IBA analog TFIBA inhibited IBA formation in leaves after application to the root system, but was not transported from roots to shoots. AM hyphae were also not able to transport TFIBA. Our results indicate complex control mechanisms to regulate the levels of free and conjugated auxins, which are locally and systemically induced during early stages of the formation of an arbuscular mycorrhizal symbiosis.     

植物乙酰辅酶A羧化酶的分子生物学与基因工程

植物中的乙酰辅酶A羧化酶(acetylCoAcarboxylase,ACCase)分两种类型:原核类型的ACCase位于质体中,是脂肪酸合成途径中的关键酶;真核类型的ACCase位于胞质溶胶中,催化形成的产物主要用于长链脂肪酸的合成以及类黄酮等次生代谢产物的合成。但禾本科植物的质体和胞质溶胶中的ACCase都属于真核类型,其中质体中的是环己烯酮类和芳氧苯氧丙酸类等除草剂作用的靶蛋白。文中主要综述了植物中ACCase的生理功能、分子生物学特征及其对两类除草剂的敏感性,并对其基因工程作了展望。     

The purification and function of acetyl coenzyme A:acyl carrier protein transacylase

When individual enzyme activities of the fatty acid synthetase (FAS) system were assayed in extracts from five different plant tissues, acetyl-CoA:acyl carrier protein (ACP) transacylase and beta-ketoacyl-ACP synthetases I and II had consistently low specific activities in comparison with the other enzymes of the system. However, two of these extracts synthesized significant levels of medium chain fatty acids (rather than C16 and C18 acid) from [14C]malonyl-CoA; these extracts had elevated levels of acetyl-CoA:ACP transacylase. To explore the role of the acetyl transacylase more carefully, this enzyme was purified some 180-fold from spinach leaf extracts. Varying concentrations of the transacylase were then added either to spinach leaf extracts or to a completely reconstituted FAS system consisting of highly purified enzymes. The results suggested that: (a) acetyl-CoA:ACP transacylase was the enzyme catalyzing the rate-limiting step in the plant FAS system; (b) increasing concentration of this enzyme markedly increased the levels of the medium chain fatty acids, whereas increase of the other enzymes of the FAS system led to increased levels of stearic acid synthesis; and (c) beta-ketoacyl-ACP synthetase I was not involved in the rate-limiting step. It is suggested that modulation of the activity of acetyl-CoA:ACP transacylase may have important implications in the type of fatty acid synthesized, as well as the amount of fatty acids formed.     

Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.     

Initiation of lipogenic enzyme activities in rat mammary glands.

The activities of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthetase remained low until parturition at 22 days of gestation and increased significantly within 1 day post partum. Administration of progesterone on days 20 and 21 and at parturition abolished the increases for at least 48 h after parturition. Removal of the pups of normal rats prevented the increases in activities of acetyl-CoA carboxylase and ATP citrate-lyase, but not of fatty acid synthetase, and administration of prolactin corticosterone or insulin did not stimulate activity. Tissue from suckled glands in which the ducts had been ligated at parturition showed no increase in the activities of acetyl-CoA carboxylase and ATP citrate-lyase within 24 h, whereas fatty acid synthetase activity was similar to that in the sham-operated contralateral glands. Foetoplacentectomy on day 18 increased the activity of fatty acid synthetase but not of acetyl-CoA carboxylase and ATP citrate-lyase; suckling of these dams by foster pups increased both acetyl-CoA carboxylase and ATP citrate-lyase.     

Estrogen regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and acetyl-CoA carboxylase in xenopus laevis

Administration of estradiol-17 beta to male Xenopus laevis evokes the proliferation of the endoplasmic reticulum and the Golgi apparatus and the synthesis and secretion by the liver of massive amounts of the egg yolk precursor phospholipoglycoprotein, vitellogenin. We have investigated the effects of estrogen on three key regulatory enzymes in lipid biosynthesis, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the major regulatory enzyme in cholesterol and isoprenoid synthesis, and acetyl-CoA carboxylase and fatty acid synthetase, which regulate fatty acid biosynthesis. HMG-CoA reductase activity and cholesterol synthesis increase in parallel following estrogen administration. Reductase activity in estrogen stimulated Xenopus liver cells peaks at 40-100 times the activity observed in control liver cells. The increased rate of reduction of HMG-CoA to mevalonic acid is not due to activation of pre-existing HMG-CoA reductase by dephosphorylation, as the fold induction is unchanged when reductase from control and estrogen-stimulated animals is fully activated prior to assay. The estrogen-induced increase of fatty acid synthesis is paralleled by a 16- to 20-fold increase of acetyl-CoA carboxylase activity, indicating that estrogen regulates fatty acid synthesis at the level of acetyl-CoA carboxylase. Fatty acid synthetase activity was unchanged during the induction of fatty acid biosynthesis by estrogen. The induction of HMG-CoA reductase and of acetyl-CoA carboxylase by estradiol-17 beta provides a useful model for regulation of these enzymes by steroid hormones.