Characterization of Fatty Acid biosynthesis in isolated pea root plastids

Fatty acid biosynthesis from Na[1-¹⁴C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl₂, 1 millimolar each of MnCl₂ and glycerol-3-phosphate, 15 millimolar KHCO₃, 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35°C. At the standard incubation temperature of 25°C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [¹⁴C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.     

Fatty acid synthesis in isolated leucoplasts of developing seeds of <Emphasis Type="Italic">Brassica campestris</Emphasis> L

Under in vitro conditions, the fatty acid synthesis from labelled substrates was studied in the leucoplasts isolated from developing seeds of Brassica campestris L. The rate of fatty acid synthesis with Na-(1-¹⁴C) acetate was higher at lower concentrations (up to 1 mM). However, with ¹⁴C(U)-D-glucose, the rate was higher at higher concentrations (3–4 mM) at all the three stages of seed development. ATP and NAD(P)H were absolutely required in acetate utilization. Even for glucose utilization, the exogenous supply of ATP and NAD(P)H was required. At the early stage of seed development, the maximum reduction in labelled glucose and acetate utilization for fatty acid synthesis was observed with pyruvate and glucose, respectively. However, at mid-early and mid-late stages, maximum reduction in their utilization for fatty acid synthesis was observed with glc-6-P. This suggests a shift in the utilization of substrates for fatty acid synthesis during the development of seeds probably via different translocators activated at different stages.     

Malate- and pyruvate-dependent Fatty Acid synthesis in leucoplasts from developing castor endosperm

Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoll gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl-CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction.     

A microsomal fatty acid synthetase coupled to acyl-CoA reductase in Euglena gracilis

Microsomal particles from dark-grown Euglena gracilis incorporated malonyl-CoA into fatty acids and fatty alcohols in the presence of acetyl-CoA, NADH, NADPH, and ATP with an optimum pH of 8.0. Schmidt degradation of the individual fatty acids derived from [l,3-¹⁴C]malonyl-CoA showed that the microsomal fatty acid synthesis was a de novo type. Detailed analysis of the products formed in the absence of various cofactors showed that the role of ATP was specifically in the formation of fatty alcohols and that fatty acid reduction specifically required NADH.The major aliphatic chains synthesized by the microsomes were C₁₆, C₁₈, and C₁₄ in both the acyl portions and alcohols. Although relative concentrations of acetyl-CoA and malonyl-CoA influenced the chain length distribution of products, C₁₆remained the major product in both the alcohol and the acid fractions. Effects of NADPH and NADH concentrations on malonyl-CoA incorporation suggested that the two reductive steps involved in the microsomal fatty acid synthesis have different pyridine nucleotide specificity. The apparent K_m for malonyl-CoA was 4.2 × 10^?4m. Based on the experimental results a mechanism is suggested by which carbon is channeled into wax esters under conditions of nutritional abundance in dark-grown E. gracilis.     

Intramyelinic conversion of cerebrosides into acylgalactosylceramides

Acylgalactosylceramide (AGC) synthesis was measured in vivo, and in a cell free system. 24 hours post-injection of [³H]palmitic acid into rat brain, more than 60% of the AGC radioactivity was associated with an ester linkage. Isolated rat myelin was incubated in the presence of [¹⁴C]palmitic acid, 2mM ATP, 50 M CoA and 10 mM MgCl₂ and acylation of myelin cerebrosides occurred at a linear rate for at least 60 min. Incubation of isolated myelin under standard conditions with [³H] cerebrosides and [¹⁴C]palmitic acid produced double labeled AGC. Labeling of AGC was maximum at pH 7.5 and 37°C and appeared to be enzyme mediated inasmuch as it was reduced by myelin incubation with trypsin and drastically reduced by preheating the myelin for 5 min at 80°C. Omission of ATP, CoA, MgCl₂ or all three did not reduce fatty acid incorporation into AGC when compared to the values in the complete system. Addition of Triton X100 or Sodium Dodecyl Sulfate had little or no effect on the acylation of cerebrosides. Pulse chase experiments indicated that the reaction involved the net addition of fatty acid to the cerebrosides, rather than a rapid fatty acid exchange.     

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-¹⁴C]malonyl-CoA was incorporated into fatty acids of C₁₆C₂₆. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C₁₂C₂₄) elongation showed that stearoyl-CoA, in the presence of [2-¹⁴C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C₂₀C₂₆. [1-¹⁴C]C₁₆CoA and [1-¹⁴C]C₁₈CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-¹⁴C]C₁₈CoA was a better primer than [1-¹⁴C]C₁₆CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-¹⁴C]C₁₈CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-¹⁴C]malonyl-CoA in the presence of C₁₈CoA, on the one hand, and [1-¹⁴C]C₁₈CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.     

Fat Metabolism in Higher Plants XXXVI: Long Chain Fatty Acid Synthesis in Germinating Peas

A low lipid, high starch containing tissue, namely cotyledons of germinating pea seedlings was examined for its capacity to synthesize fatty acid. Intact tissue slices readily incorporate acetate-¹⁴C into fatty acids from C₁₆ to C₂₄. Although crude homogenates synthesize primarily 16:0 and 18:0 from malonyl CoA, subsequent fractionation into a 10,000g pellet, a 10⁵g pellet and supernatant (soluble synthetase) revealed that the 10⁵g pellet readily synthesizes C₁₆ to C₂₈ fatty acids whereas the 10,000g and the supernatant synthesize primarily C₁₆ and C₁₈. All systems require acyl carrier protein (ACP), TPNH, DPNH if malonyl CoA is the substrate and ACP, Mg²⁺, CO₂, ATP, TPNH, and DPNH if acetyl CoA is the substrate. The cotyledons of germinating pea seedlings appear to have a soluble synthetase and 10,000g particles for the synthesis of C₁₆ and C₁₈ fatty acid, and 10⁵g particles which specifically synthesize the very long chain fatty acid from malonyl CoA, presumably via malonyl ACP.     

Studies on the uptake of fatty acids by Escherichia coli

Oleate uptake by Escherichia coli showed saturation kinetics with a K_m of 34 μm and an activation energy of 6.25 kcal/mole indicating that the rate limiting step in oleate uptake involves an enzyme-catalyzed step. The rate of oleate uptake was decreased by the respiratory poisons, arsenate and 4-pentenoate, which apparently is activated to pentenoyl CoA, thus reducing the intracellular concentration of free intracellular CoA. These data indicated that oleate uptake is dependent on cellular ATP and CoA. During short pulses with [1-¹⁴C]oleate, most of the radioactivity which was taken up was released as ¹⁴C0₂; cells accumulated radioactivity in phospholipids and compounds with the chromatographic mobility of Krebs cycle intermediates. Neither free fatty acid nor oleyl CoA were detectable in the cells. The results support the hypothesis that long-chain fatty acids are translocated by the long-chain fatty acyl CoA synthetase and that uptake is the rate limiting step in the utilization of exogenous fatty acid.     

Biosynthesis of Fatty acids by a soluble extract from developing soybean cotyledons

Fractionation of developing soybean cotyledons into cellular components demonstrates that most of the activity necessary to incorporate acetate-1-¹⁴C into lipid remains in the supernatant from a 198,000g spin for 1 hr. The system studied is dependent upon ATP, CoA, and CO₂. Concentrations of ATP greater than 4 × 10⁻³m are inhibitory, while 1 × 10⁻⁴m CoA is needed for optimal activity. Avidin inhibition of acetate incorporation into lipid could be reversed by biotin. Studies indicated that NADPH is a better source of reducing power than NADH. The system studied is inhibited by p-chloromercuribenzoic acid and this inhibition can be reversed by an excess of GSH. The system studied shows maximum activity in tris buffer at pH 8.6 or in glycine buffer, pH 9.4.     

Rates and products of long-chain Fatty Acid synthesis from [1-C]acetate in chloroplasts isolated from leaves of 16:3 and 18:3 plants

Chloroplasts highly active in the synthesis of long-chain fatty acids from [1-¹⁴C]acetate were prepared from leaves of Solanum nodiflorum, Chenopodium quinoa, Carthamus tinctorius, and Pisum sativum. These preparations were used to test whether the various additions to incubation media found to stimulate the synthesis of particular lipid classes in vitro by Spinacia oleracea chloroplasts were applicable generally. Chloroplasts from 18:3 plants incorporated a greater proportion of radioactivity into unesterified fatty acids under control conditions than did those from 16:3 plants. Supplying exogenous sn-glycerol 3-phosphate or Triton X-100 to chloroplasts increased the synthesis of glycerolipids in all cases and accentuated the capacity of chloroplasts from 18:3 plants to accumulate phosphatidic acid rather than the diacylglycerol accumulated by chloroplasts from 16:3 plants. The UDP-galactose-dependent synthesis of labeled diacylgalactosylglycerol was much less active in incubations of chloroplasts from 18:3 plants also containing sn-glycerol 3-phosphate and Triton X-100 compared with similar incubations from 16:3 plants. Exogenous CoA stimulated total fatty acid synthesis in all chloroplast preparations and the further addition of ATP diverted radioactivity from the unesterified fatty acid to acyl-CoA. The results have been discussed in terms of the two pathway hypothesis for lipid synthesis in leaves.     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

An in vitro system of autotropic synthesis of activated acetic acid from¹⁴CO₂ inMethanobacterium thermoautotrophicum was developed.

1. A recognized¹⁴CO₂-fixation product in vitro was activated [¹⁴C] acetic acid. It could be trapped enzymatically into citrate and released again as [¹⁴C] acetate by citrate synthase and citrate lyase, respectively.
2. The synthesis of both activated acetic acid and methane from CO₂ proceeded in parallel under a variety of conditions. Both of these processes were stimulated greatly and to the same extent by the addition of methyl coenzyme M to the assay.
3. Various inhibitors of methanogenesis tested also inhibited acetate synthesis, e.g. CH₂Cl₂, CHCl₃, CCl₄, N₂O, and bromoethane sulfonic acid. Cyanide specifically inhibited the synthesis of activated acetic acid, whereas methane formation was unaffected. Cyanide inhibition was relieved by adding CO, whereas the inhibition by the other compounds was not.

The data suggest: The product studied in vitro was acetyl CoA. Its synthesis involves intermediates of CO₂ reduction to methane. In addition, a cyanide-sensitive reaction is required which does not participate in CO₂ reduction to methane.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

Biosynthesis of lipids in chloroplasts isolated from jack pine needles

Suspensions of isolated pine needle chloroplasts were shown to incorporate galactose from UDP galactose-[¹⁴C] into galactolipids. The incorporation of the label among galactolipids was always considerably higher in the monogalactosyl diglycerides than in the digalactosyl diglycerides. The galactosyl incorporation into both galactolipid fractions was optimal at pH 8.0 and was inhibited by sulphydryl reagents (p-chloromercuribenzoate, N-ethyl maleimide and CdCl₂). The chloroplast preparations were also able to biosynthesize various phospholipids and galactolipids from palmitoyl-[1-¹⁴C]-CoA; the major portion of the label appeared in phosphatidyl choline. The incorporation of palmitic-[1-¹⁴C] acid into various lipids was very poor compared to that of palmitoyl-[1-¹⁴C]-CoA. However, addition of ATP and CoA markedly stimulated lipid biosynthesis from palmitic-[1-¹⁴C] acid, suggesting the presence of activating enzymes. These chloroplast suspensions did not show any de novo fatty acid synthesis.     

Localisation and characterization of the fatty acid synthesizing system in cells of Glycine max (soubean) suspension cultures

In course of a study of fatty acid synthetase in higher plants, non-green cell suspension cultures of Glycine max (soybean) served as model tissues. For the first time, a fatty acid synthesizing system was characterized in cell cultures of higher plants and was found to be solely located in proplastids of the cells. Optimum activity of the fatty acid synthesizing system in proplastids was observed between pH 8.0 and 8.2; with [1-14C]acetate as substrate, cofactors required were CoA, ATP, Mn2+, Mg2+, HCO3-, NADH and NADPH. The system was more sensitive towards NADH than NADHP. [1-14C]Acetate,[2-14C]-malonate and [3-14C]pyruvate served as precursors for fatty acids, indicating the presence of pyruvate dehydrogenase activity in proplastids. In disrupted proplastids, [2-14C]malonylCoA was a better precursor than [1-14C]acetylCoA. After incubation of proplastids with [2-14C]malonate, a small shift, from palmitic acid to higher homologs, of label incorporated was observed, as compared to incorporation of label from [1-14C]acetate and [3-14C]pyruvate. Under the conditions of the experiment, only small amounts of polyunsaturated fatty acids, the main fatty acid components of this organelle, were synthesized. In respect to fatty acid synthesis, the non-green cell suspension culture resembles photosynthetic leaf tissue.     

Preincubation stimulates fatty acid synthesis by liver slices and isolated hypatocytes from fasted and diabetic rats.

We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1-¹⁴C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10^?4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1-¹⁴C] acetate into fatty acids or cholesterol. The rate of ¹⁴CO₂ synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1-¹⁴C] acetate or [³H] H₂O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

Flavaspidic acid: a biochemical probe for the study of metabolic pathway in hepatocytes.

Displacement of fatty acids or their CoA esters from the cytosolic fatty acid-binding-protein (FABP) by β-flavaspidic acid-N-methyl-glucaminate (flavaspidic acid) shifted the flow of lactate from the lipogenic to the gluconeogenic pathway in hepatocytes isolated from livers of fed rats. Analyses of radiolabel in products of 1 hour incubations utilizing 40 mM DL-sodium lactate (U-¹⁴C sodium lactate or ³H₂O) as substrate showed fatty acid synthesis to be depressed in the presence of 5 mM flavaspidic acid to levels ranging to 28% of control. Glucose synthesis, on the other hand, rose to a level 809% of control, a rate approximately equivalent to the maximal rate of gluconeogenesis found in hepatocytes from diabetic rats. The production of ketone bodies was not influenced by flavaspidic acid whereas CO₂ production decreased reflecting the diversion of the flow of lactate from the lipogenic to the gluconeogenic pathway.These results are presented in support of the hypothesis that unbound long-chain acyl CoA esters act as physiological effectors of the adenine nucleotide translocation and through this action, integrate lipogenic and gluconeogenic activities. Flavaspidic acid thus serves as a useful probe for studies of the role of long chain acyl CoA esters in the intracellular regulation of intermediary metabolism and of the modulation exerted by the FABP.     

Fat metabolism in higher plants: The nonenzymatic acylation of dithiothreitol by acyl coenzyme A

A compound soluble in organic solvents and synthesized from [¹⁴C]acetate by isolated spinach chloroplasts incubated in the dark in the presence of dithiothreitol was shown to be O-acetyl dithiothreitol. The chloroplast system was required for the activation of acetate to acetyl CoA, but the transfer of the acetyl moiety to dithiothreitol was nonenzymatic. The first product of the reaction was shown to be S-acetyl dithiothreitol, but in the presence of an oxidant, simultaneous ring closure and migration of the acetyl group from the thiol to an adjacent hydroxyl group occurred to form an O-acetyl dithiothreitol.The acetyl transfer reaction involving acetyl CoA and dithiothreitol showed a marked pH dependence, being most active at about pH 9 and inoperative below pH 6. All acyl CoAs tested (C₂-C₁₈) rapidly labeled dithiothreitol; acetyl acyl carrier protein, and palmityl acyl carrier protein were much less reactive and free fatty acids were unreactive. The thiol reagents dithioerythritol, glutathione, and cysteine, in addition to dithiothreitol, reacted rapidly with acetyl CoA to form the corresponding acetyl mercaptans. 2-Mercaptoethanol was much less reactive; oxidized dithiothreitol was unreactive. The second-order rate constant for acetyl dithiothreitol synthesis was 12.3 m_?1 min^?1 at pH 8.5 and 30 °C.     

A novel direct homogeneous assay for ATP citrate lyase

ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg²⁺ as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [¹⁴C]acetyl-CoA without detecting [¹⁴C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1) incubation of ACL enzyme with [¹⁴C]citrate and other substrates/cofactors CoA, ATP, and Mg²⁺, 2) EDTA quench, 3) addition of MicroScint-O, the agent that specifically detects product [¹⁴C]acetyl-CoA, and 4) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.     

Long chain fatty acid synthesis in developing castor bean seeds I. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system

The path of LFA synthesis from acetate in developing castorbean seeds was associated with subcellular 10,000 g particles.Further fractionation of these particles by a stepwise densitygradient method showed the high possibility that the site ofLFA synthesis is the proplastid. A study on cofactor requirementswhen [1-¹⁴C]acetate predominantly incorporated into LFAs indicatedthat synthesis would be achieved by acetyl-CoA carboxylation,malonyl-ACP condensation. ATP, CoA, HCO₃^– and Mg⁺⁺ orMn⁺⁺ were essential for synthesis from acetate by the 10,000gparticulate system. Results of inhibhitor experiment suggestedthat the supply of ATP to the LFA synthesizing system is broughtabout by mitochondrial oxidative phosphorylation, when acetateis the sole precursor for LFA synthesis in this system. TheNADPH generating system was contained in the paticles, althoughthe addition of NADP⁺ and G-6-P increased synthesis. NADH markedlystimulated LFA synthesis from acetate. The primary role of NADHseems to be as a direct reductant in both steps involving thereduction and oxidative desaturation of fatty acid chains; particularly,in the former step, although NADH partially contributes to thesupply of ATP as a respiratory substrate. It is unlikely thatNADH works as a hydrogen donor to NADP⁺. LFA synthesis by thecastor bean particulate system was not stimulated by light,thus differing from that by leaf chloroplasts. (Received July 23, 1973; )