Determination of Acetyl-CoA and Malonyl-CoA in Germinating Rice Seeds Using the LC-MS/MS Technique

A highly sensitive and selective analytical method was developed to determine levels of acetyl-CoA and malonyl-CoA in plant tissues. The analytical method includes a convenient extraction method for plant samples and a new LC-MS/MS technique utilizing an ion pair reagent. These acyl-CoAs, present in germinating rice seeds, were then determined by the method developed. It was found that the concentrations of both acetyl-CoA and malonyl-CoA increased with time during the germination of rice seeds and also increased at the elevated cultivation temperatures among tested. These results reflect the development of enzymes that produce these acyl-CoAs in germinating rice seeds.     

Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS

Malonyl-CoA is a key intermediate involved in lipid synthesis and lipid oxidation. Here, we report on a novel method for the quantification of malonyl-CoA and seven other short-chain acyl-CoAs in various rat and mouse tissues using ion-pairing reversed-phase HPLC/MS. This method is capable of measuring malonyl-CoA, free coenzyme A (CoASH), acetyl-CoA, beta-hydroxyl-butyryl-CoA (HB-CoA), 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), propionyl-CoA, succinyl-CoA, and isobutyryl-CoA simultaneously with a dynamic linear range over two orders of magnitude in a 7.0 min HPLC gradient run. The lower limit of quantification (LLOQ) was 0.225 pmol for all acyl-CoAs studied, except for HMG-CoA which had a higher LLOQ of 0.90 pmol. The interference of HB-CoA on the quantification of malonyl-CoA in animal tissues was also explored for the first time.     

Assay of long-chain acyl-CoAs in a complex reaction mixture

A method has been developed which allows the quantitative analysis of labeled or unlabeled acyl-CoAs in complex reaction mixtures. The method is based on (a) a quantitative solubilization of acyl-CoAs and lipids, directly in the reaction vessel, by 0.05 M Tris-HCl, pH 7.5/CHCl3/CH3OH (1/3/3, v/v/v); (b) monodimensional TLC of aliquots of the whole reaction mixture, resolving malonyl-CoA, acetyl-CoA, long-chain acyl-CoAs, polar lipids and neutral lipids plus free fatty acids; and (c) quantitation by TLC densitometry and/or TLC radiochromatography. All fractions--and particularly long chain-acyl-CoAs--can then be analyzed for distribution and label of fatty acyl moieties.     

Peroxisomal fatty acid oxidation is a substantial source of the acetyl moiety of malonyl-CoA in rat heart

Little is known about the sources of acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of mitochondrial fatty acid oxidation in the heart. In perfused rat hearts, we previously showed that malonyl-CoA is labeled from both carbohydrates and fatty acids. This study was aimed at assessing the mechanisms of incorporation of fatty acid carbons into malonyl-CoA. Rat hearts were perfused with glucose, lactate, pyruvate, and a fatty acid (palmitate, oleate or docosanoate). In each experiment, substrates were (13)C-labeled to yield singly or/and doubly labeled acetyl-CoA. The mass isotopomer distribution of malonyl-CoA was compared with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of labeled glucose or lactate/pyruvate, the (13)C labeling of malonyl-CoA was up to 2-fold lower than that of mitochondrial acetyl-CoA. However, in the presence of a fatty acid labeled in its first acetyl moiety, the (13)C labeling of malonyl-CoA was up to 10-fold higher than that of mitochondrial acetyl-CoA. The labeling of malonyl-CoA and of the acetyl moiety of citrate is compatible with peroxisomal beta-oxidation forming C(12) and C(14) acyl-CoAs and contributing >50% of the fatty acid-derived acetyl groups that end up in malonyl-CoA. This fraction increases with the fatty acid chain length. By supplying acetyl-CoA for malonyl-CoA synthesis, peroxisomal beta-oxidation may participate in the control of mitochondrial fatty acid oxidation in the heart. In addition, this pathway may supply some acyl groups used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes.     

Biosynthesis of fatty acids in mouse brain mitochondria in the presence of malonyl-CoA or acetyl-CoA]

Incorporation of malonyl-CoA or acetyl-CoA is studied in mouse brain mitochondrial fatty acids. Rupture of mitochondria is necessary ; Triton X-100 gives the best result. Other detergents or sonication are of lesser efficiency. Cofactor requirements have been studied : NADH and NADPH have been tested ; ATP increases biosynthesis and CoA causes an inhibition. Two systems of biosynthesis are involved : -- One is a de novo system using malonyl-CoA. Malonyl-CoA alone is incorporated and synthesizes mainly C16, indicating the existence of a malonly-CoA decarboxylase although elongation of short chain fatty acids cannot be excluded. Addition of acetyl-CoA increases the biosynthesis and palmityl-CoA when added causes an inhibition. -- The other system, using acetyl-CoA, elongates exogenous palmityl-CoA ; endogenous acyl-CoAs are not elongated by acetyl-CoA. All these results are confirmed by radiogas chromatographic studies of the reactions products.     

Regulation of lipid synthesis in soybeans by two benzoic Acid herbicides

The effects of 3-nitro-2,5-dichlorobenzoic acid (dinoben) and 3-amino-2,4-dichlorobenzoic acid (chloramben) on lipid formation and on the incorporation of various substrates into lipids by intact seeds and subcellular fractions of germinating soybean (Glycine max [L.] Merr. ;Amsoy') were studied. Dinoben (20 mug/ml) inhibited synthesis of total lipids 67%, neutral lipids 73%, glycolipids 51%, and phospholipids 39% in germinating seeds. When polar lipids were analyzed further, inhibition of individual lipid classes was also observed. Chloramben (20 mug/ml) stimulated total lipid synthesis 25%. With the exception of the mitochondrial fraction where malonate thiokinase was absent, dinoben inhibited up to 99% the incorporation of acetate and malonate into lipids, but did not inhibit acetyl-CoA and malonyl-CoA incorporation. Chloramben stimulated the incorporation of all substrates tested into lipids by all fractions except the mitochondrial fraction when malonate was the substrate. When dinoben and chloramben were used in combinations, chloramben did not reverse the inhibitory effect of dinoben.It is concluded that the dinoben inhibitory effect is specific and is associated with the acetate and malonate thiokinase systems. The chloramben effect is stimulatory to either acetyl-CoA carboxylase or fatty acid synthetase or both.     

Subcellular distribution of acetyl-coenzyme A carboxylase in mesophyll cells of barley and sorghum leaves

The subcellular distribution of acetyl-CoA carboxylase [acetyl-CoA-carbon dioxide ligase (ADP-forming), EC 6.4.1.2] was determined in mesophyll protoplasts isolation from barley, a C3 plant, and sorghum, a C4 plant. In both species, all of the mesophyll acetyl-CoA carboxylase was demonstrated to be chloroplastic. In barley leaves and mesophyll protoplasts, a single biotinyl protein of 60,000 Da was identified by a modified Western-blotting procedure. The subcellular distribution of this biotinyl protein was identical to that found for acetyl-CoA carboxylase. These results are discussed in relation to the compartmentation of reactions requiring malonyl-CoA as a substrate.     

Fatty acid synthetase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from pigeon liver.

The kinetic mechanism of pigeon liver fatty acid synthetase action has been studied using steady state kinetic analysis. Initial velocity studies are consistent with an earlier suggestion that the enzyme catalyzes this reaction by a seven-site ping-pong mechanism. Although the range of substrate concentrations that could be used was limited by several factors, the initial velocity patterns showing the relationship between the substrates acetyl coenzyme CoA, malonyl-CoA, and NADPH appear to be a series of parallel lines, regardless of which substrate is varied at fixed levels of a second substrate. However, two of the substrates, acetyl-CoA and malonly-CoA, apparently exhibit a competitive substrate inhibition with respect to each other, but NADPH shows no inhibition of any kind. Product inhibition patterns suggest that free CoA is competitive versus acetyl-CoA and malonyl-CoA and is uncompetitive versus NADPH, and that NADP+ is competitive versus NADPH and uncompetitive versus acetyl-CoA or malonyl-CoA. These results are consistent with a seven-site ping-pong mechanism with intermediates covalently bound to 4'-phosphopantetheine (part of acyl carrier protein). Double competitive substrate inhibition by acetyl-CoA and malonyl-CoA is consistent with the rate equation derived for the over-all mechanism. The kinetic mechanism developed from these results is capable of explaining the formation of fatty acids from malonyl-CoA and NADPH alone (Katiyar, S. S., Briedis, A. V., and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420) and also the formation of triacetic acid lactone from either malonyl-CoA alone or acetyl-CoA plus malonyl-CoA.     

Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion.

Several approaches were used to test the hypothesis proposing a role for acyl-CoA esters in nutrient-induced insulin release (Prentki, M., and Matschinsky, F. M. (1987) Physiol. Rev. 67, 1185-1248; Corkey, B. E., Glennon, M. C., Chen, K. S., Deeney, J. T., Matschinsky, F. M., and Prentki, M. (1989) J. Biol. Chem. 264, 21608-21612). Exogenous saturated long chain fatty acids markedly potentiated glucose-induced insulin release and elevated long chain acyl-CoA esters in the clonal beta-cell line (HIT). The secretory action depended on the fatty acid chain length, occurred in the range 3-20 microM (free concentration of palmitate), and was reversible and inhibitable by the neuromodulator somatostatin. 2-Bromopalmitate, an inhibitor of carnitine palmitoyl transferase I, suppressed the oxidation of endogenous fatty acids and promoted release of insulin. Only the nutrients or the combination of nutrients that caused secretion elevated malonyl-CoA. The short-chain acyl-CoA profile of HIT cells stimulated by various nutrients was determined in the presence of the nonstimulatory fuel glutamine. Glucose and leucine each provoked similar changes in acyl-CoA compounds. Both secretagogues elevated malonyl-CoA 3-6-fold, whereas succinyl-CoA, free CoASH, acetyl-CoA, and the free CoASH to acetyl-CoA ratio remained unaltered. Furthermore, only when inhibition of fatty acid oxidation was associated with a rise in malonyl-CoA did the total (mitochondrial plus cytoplasmic) content of long chain acyl-CoA esters correlate inversely with insulin release promoted by various nutrients. The results are consistent with the concept that fuel stimuli cause a rise in malonyl-CoA which by inhibiting fatty acid oxidation increase cytosolic long chain acyl-CoA esters. These data provide further support for a model in which malonyl-CoA and long chain acyl-CoAs esters serve as metabolic coupling factors when pancreatic beta-cells are stimulated with glucose and other nutrient secretagogues.     

Changes in the size and composition of intracellular pools of nonesterified coenzyme A and coenzyme A thioesters in aerobic and facultatively anaerobic bacteria.

Intracellular levels of three coenzyme A (CoA) molecular species, i.e., nonesterified CoA (CoASH), acetyl-CoA, and malonyl-CoA, in a variety of aerobic and facultatively anaerobic bacteria were analyzed by the acyl-CoA cycling method developed by us. It was demonstrated that there was an intrinsic difference between aerobes and facultative anaerobes in the changes in the size and composition of CoA pools. The CoA pools in the aerobic bacteria hardly changed and were significantly smaller than those of the facultatively anaerobic bacteria. On the other hand, in the facultatively anaerobic bacteria, the size and composition of the CoA pool drastically changed within minutes in response to the carbon and energy source provided. Acetyl-CoA was the major component of the CoA pool in the facultative anaerobes grown on sufficient glucose, although CoASH was dominant in the aerobes. Therefore, the acetyl-CoA/CoASH ratios in facultatively anaerobic bacteria were 10 times higher than those in aerobic bacteria. In Escherichia coli K-12 cells, the addition of reagents to inhibit the respiratory system led to a rapid decrease in the amount of acetyl-CoA with a concomitant increase in the amount of CoASH, whereas the addition of cerulenin, a specific inhibitor of fatty acid synthase, triggered the intracellular accumulation of malonyl-CoA. The acylation and deacylation of the three CoA molecular species coordinated with the energy-yielding systems and the restriction of the fatty acid-synthesizing system of cells. These data suggest that neither the accumulation of acetyl-CoA nor that of malonyl-CoA exerts negative feedback on pyruvate dehydrogenase and acetyl-CoA carboxylase, respectively.     

Formation of mevalonic acid, sterols and bile acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in the liver of rabbits with experimental hypercholesterolemia

The effect of cholesterol diet on the rate of mevalonic acid biosynthesis from 1-14C acetyl-CoA, 2-14C malonyl-CoA and the incorporation of these substrates into sterols and bile acids in rabbit liver were studied. Simultaneously, the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and the biosynthesis of fatty acids from acetyl-CoA and malonyl-CoA were measured. Hypercholesterolemia was found to be concomitant with the inhibition of acetyl-CoA carboxylase activity only in cell-free (700 g) and mitochondrial fractions and slightly decreased the incorporation of acetyl-CoA and malonyl-CoA into fatty acids in the postmitochondrial fraction. The HMG-CoA reductase activity in all subcellular fractions except for the postmicrosomal one was inhibited under these conditions. A significant decrease of acetyl-CoA incorporation and an increase in malonyl-CoA incorporation into mevalonic acid in all liver fractions except for microsomal one were observed in rabbits with hypercholesterolemia. These data provide evidence for the existence of two pathways of mevalonic acid synthesis from the above-said substrates that are differently sensitive to cholesterol. Cholesterol feeding resulted in a decreased synthesis of the total unsaponified fraction including cholesterol from acetyl-CoA, malonyl-CoA and mevalonic acid. The rate of incorporation of these substrates into lanosterol was unchanged. All the indicated substrates (acetyl-CoA, malonyl-CoA, mevalonic acid) are precursors of bile acid synthesis in rabbit liver. Cholesterol feeding and the subsequent development of hypercholesterolemia resulted in bile acid synthesis stimulation, preferentially in the formation of the cholic + deoxycholic acids from these precursors.     

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-¹⁴C]18:0)-CoA was synthesized from [1-¹⁴C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-¹⁴C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-¹⁴C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [¹⁴C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.     

Plant regeneration from protoplasts of indica rice: genotypic differences in culture response

Summary Fourteen varieties of indica rice (Oryza saliva L.) were examined for their capacity for plant regeneration from protoplasts using the nurse culture methods developed for japonica rice. Calli induced from germinating seeds were grouped into two types: type I, white and compact; type II, yellow and friable. In four varieties producing type II callus, colony formation (2%–4.5%) and plant regeneration (2%–35%) were observed. The inability to develop suspension cultures was a major obstacle in regenerating plants from protoplasts of the remaining rice varieties studied.     

Determination of Free Ammonium and Asparagine and Glutamine Amide-Nitrogen in Extracts of Plant Tissue

A relatively simple and rapid procedure for the measurement of free ammonium and the amides in plant extracts is described. The method was developed by combining a cation-exchange method for blood ammonia with a differential acid-hydrolysis procedure for asparagine and glutamine amide-nitrogen.The recovery of standard samples (100-400 mug of ammonium- or amide-nitrogen) of free ammonium, asparagine, and glutamine after being run through the extraction, column, and analytical procedures ranged between 99 and 102%.The harvest, extraction, and analytical procedures were tested on shoots from 4 to 6-day-old germinating barley seeds. The high levels of the amides and the low level of free ammonium present in the tissue extracts indicated that the extraction and analytical procedures resulted in little if any hydrolysis of the amides.     

Regulation of malonyl-CoA concentration and turnover in the normal heart

The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH(13)CO(3). This turnover is a direct measurement of the flux of acetyl-CoA carboxylation in the intact heart. It also reflects the rate of malonyl-CoA decarboxylation, i.e. the only known fate of malonyl-CoA in the heart. Conditions were selected to result in stable malonyl-CoA concentrations ranging from 1.5 to 5 nmol.g wet weight-(1). The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol.min(-) (1).g wet weight(-1) (slope = 0.98, r(2) = 0.94). The V(max) activities of acetyl-CoA carboxylase and of malonyl-CoA decarboxylase exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. However, conditions of perfusion that increased acetyl-CoA supply resulted in higher turnover and concentration, demonstrating that malonyl-CoA turnover is regulated by the supply of acetyl-CoA. The only condition where the activity of malonyl-CoA decarboxylase regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, resulting in increased malonyl-CoA concentration and decreased turnover. Our data show that, in the absence of enzyme inhibitors, the rate of acetyl-CoA carboxylation is the main determinant of the malonyl-CoA concentration in the heart.     

Fatty Acid Chain Elongation in Palmitate-perfused Working Rat Heart: MITOCHONDRIAL ACETYL-CoA IS THE SOURCE OF TWO-CARBON UNITS FOR CHAIN ELONGATION*

Rat hearts were perfused with [1,2,3,4-¹³C₄]palmitic acid (M+4), and the isotopic patterns of myocardial acylcarnitines and acyl-CoAs were analyzed using ultra-HPLC-MS/MS. The 91.2% ¹³C enrichment in palmitoylcarnitine shows that little endogenous (M+0) palmitate contributed to its formation. The presence of M+2 myristoylcarnitine (95.7%) and M+2 acetylcarnitine (19.4%) is evidence for β-oxidation of perfused M+4 palmitic acid. Identical enrichment data were obtained in the respective acyl-CoAs. The relative ¹³C enrichment in M+4 (84.7%, 69.9%) and M+6 (16.2%, 17.8%) stearoyl- and arachidylcarnitine, respectively, clearly shows that the perfused palmitate is chain-elongated. The observed enrichment of ¹³C in acetylcarnitine (19%), M+6 stearoylcarnitine (16.2%), and M+6 arachidylcarnitine (17.8%) suggests that the majority of two-carbon units for chain elongation are derived from β-oxidation of [1,2,3,4-¹³C₄]palmitic acid. These data are explained by conversion of the M+2 acetyl-CoA to M+2 malonyl-CoA, which serves as the acceptor for M+4 palmitoyl-CoA in chain elongation. Indeed, the ¹³C enrichment in mitochondrial acetyl-CoA (18.9%) and malonyl-CoA (19.9%) are identical. No ¹³C enrichment was found in acylcarnitine species with carbon chain lengths between 4 and 12, arguing against the simple reversal of fatty acid β-oxidation. Furthermore, isolated, intact rat heart mitochondria 1) synthesize malonyl-CoA with simultaneous inhibition of carnitine palmitoyltransferase 1b and 2) catalyze the palmitoyl-CoA-dependent incorporation of ¹⁴C from [2-¹⁴C]malonyl-CoA into lipid-soluble products. In conclusion, rat heart has the capability to chain-elongate fatty acids using mitochondria-derived two-carbon chain extenders. The data suggest that the chain elongation process is localized on the outer surface of the mitochondrial outer membrane.     

Evidence of a biotin dependent acetyl-coenzyme A carboxylase in rat muscle.

Rat hindlimb muscle tissue was extracted from male Sprague-Dawley rats exsanguinated under light ether anesthesia. Muscle homogenates (50,000 x g supernatant) were incubated with ATP, bicarbonate, acetyl-CoA, and citrate. The quantity of malonyl-CoA synthesized was determined by malonyl-CoA incorporation into long acyl chains using tritiated acetyl-CoA and fatty acid synthetase. Malonyl-CoA synthesis was found to be dependent on the presence of ATP, bicarbonate, citrate, and acetyl-CoA in the incubation medium. Incubation with avidin showed near complete inhibition of carboxylation that was restored with the addition of biotin. These results represent strong evidence of a biotin containing acetyl-CoA carboxylase in skeletal muscle.     

Modification of bovine kidney pyruvate dehydrogenase kinase activity by CoA esters and their mechanism of action

The activation of pyruvate dehydrogenasea kinase activity by CoA esters has been further characterized. Half-maximal activation of kinase activity was achieved with about 1.0 microM acetyl-CoA after a 20-s preincubation in the presence of NADH. More than 80% of the acetyl-CoA was consumed during this period in acetylating sites in the pyruvate dehydrogenase complex as a result of the transacetylation reaction proceeding to equilibrium. At 1.0 microM acetyl-CoA, this resulted in more than a 4-fold higher level of CoA than residual acetyl-CoA. Activation of kinase activity could result either from acetylation of specific sites in the complex or tight binding of acetyl-CoA. Removal of CoA enhanced both acetylation and activation, suggesting acetylation mediates activation. For allosteric binding of acetyl-CoA to elicit activation, an activation constant, Ka, less than 50 nM would be required. To further distinguish between those mechanisms, the effects of other CoA esters as well as the reactivity of most of the effective CoA esters were characterized. Several short-chain CoA esters enhanced kinase activity including (in decreasing order of effectiveness) malonyl-CoA, acetoacetyl-CoA, propionyl-CoA, and methylmalonyl-CoA. Butyryl-CoA inhibited kinase activity as did high concentrations of long-chain acyl-CoAs. Inhibition by long-chain acyl-CoAs may result, in part, from detergent-like properties of those esters. Malonyl-CoA, propionyl-CoA, butyryl-CoA, and methylmalonyl-CoA, obtained with radiolabeled acyl groups, were shown to acylate sites in the complex. Propionyl-CoA and butyryl-CoA were tested, in competition with acetyl-CoA or pyruvate, as alternative substrates for acylation of sites in the complex and as competitive effectors of kinase activity. Propionyl-CoA alone rapidly acylated sites in the complex at low concentrations, and low concentrations of propionyl-CoA were effective in activating kinase activity although only a relatively small activation was observed. When an equivalent level (20 microM) of acetyl-CoA and propionyl-CoA was used, marked activation of kinase activity due to a dominant effect of acetyl-CoA was associated with acetylation of a major portion of sites in the complex and with a small portion undergoing acylation with propionyl-CoA. Those results were rapidly achieved in a manner independent of the order of addition of the two CoA esters. That indicates that tight slowly reversible binding of acetyl-CoA is not involved in kinase activation. High levels of propionyl-CoA greatly reduced acetylation by acetyl-CoA and nearly prevented activation of kinase activity by acetyl-CoA.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.