Fatty acyl-CoA Derivatives Research Articles

Molecular and functional characterization of the ceramide synthase from Trypanosoma cruzi

In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [3H]dihydrosphingosine or [3H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B1, a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasite's CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B1 did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasite's CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1Δ lac1Δ double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B1 than the parental strain.

Expression of iNOS is reduced by an inhibitor of fatty acyl CoA synthetase.

ObjectiveTo test the hypothesis that fatty acyl CoA derivatives of fatty acids modulate cytokine stimulated iNOS expression in C6 astrocytes.RationaleTriacsin C, a fungal metabolite, inhibits fatty acyl CoA synthetase in many cell types. The compound inhibits fatty acid induced iNOS expression and apoptosis in isolated islets of Langerhans (Shimabukuro et al 1996), suggesting that fatty acyl CoA derivatives regulate iNOS expression.MethodsC6 cells were stimulated with a cytokine mix consisting of TNFα (60 ng/mL), ILβ (2 ng/mL) and IFNγ (100 units/mL) in the presence or absence of triacsin C. Expression of iNOS was estimated by measuring iNOS protein by immunoblotting. In vivo iNOS activity was estimated by measuring media nitrite (Greiss reaction) and in vitro iNOS activity was measured by conversion of [14C]arginine to [14C]citrulline in the absence of added calcium.ResultsIn C6 cell homogenates, triacin C inhibited FACS activity in a concentration dependent manner, ranging from 31.9 ± 1.5% at 300 nM to 71.1 ± 3.8% at 5 μM. The effect of 5 μM triacsin C on cytokine‐stimulated media nitrite, catalytic activity and immunoreactive protein were measured at 18 and 24 hours: % of Cytokine Stimulated Activity Media Nitrite Catalytic Activity Immunoreactivity 18 hours 62.5 ± 6.9% 103 ± 11.1% 25.1 ± 13.2% 24 hours 80.6 ± 1.39%* 373 ± 72%* 36.6 ± 5.81% = p< 0.05 as compared to 18 hours by Student's ttest; n=3 independent experiments for each parameter, each done in duplicate or triplicate. ConclusionsThese results suggest that triacsin C inhibits cytokine stimulated NO formation, and that iNOS protein is significantly less in triacsin C treated cells. Surprisingly, not all of the immunoreactive iNOS protein seems to be catalytically active. The data suggest that triacsin C may delay cytokine‐stimulated expression of iNOS and thus its overall activity in C6 cells.

Carnitine and type 2 diabetes

Studies in humans and animals demonstrate that "lipid over supply" causes or worsens insulin resistance via multiple mechanisms involving the accumulation of intracellular lipids in multiple tissues. In particular, the accumulation of fatty acyl CoA derivatives/metabolites in muscle inhibits both insulin signaling and glucose oxidation. Therefore agents that ameliorate the accumulation of fatty acyl CoA derivatives and/or their metabolites would be beneficial in the treatment or prevention of insulin resistance and T2D. Hyperinsulemic/euglycemic clamp studies in humans and carnitine supplementation studies in rodents provide "proof-of-concept" that carnitine is effective at improving insulin-stimulated glucose utilization and in reversing abnormalities of fuel metabolism associated with T2D. Carefully controlled clinical trials are warranted to determine the efficacy dietary carnitine supplementation as an adjunctive treatment for type 2 diabetes.

Adaptation and Maladaptation of the Heart in Obesity

Obesity affects the cardiovascular system at many different levels, including the heart muscle itself. Clinical and experimental studies have shown an accumulation of triglycerides and other lipid species in cardiomyocytes. Analogous to hepatic steatosis, investigators have introduced the term “cardiac steatosis”. The present review addresses the complex relationships between cardiac fuel homeostasis, insulin resistance, and proposed mechanisms of damage to cardiomyocytes in different models of obesity, insulin resistance, and lipotoxicity. Specifically, the review weighs the evidence whether there is a heart muscle disorder in human obesity. It discusses how adipokines can modulate cardiac metabolism, and it focuses on the metabolic remodeling accompanying increased fatty acid supply in the heart of rodent models of lipotoxicity, with special attention to the role played by mitochondrial uncoupling and futile cycling. We stress the notion that, in spite of the many proposed mechanisms, cardiac lipotoxicity is still a hypothesis rather than an established pathophysiologic principle. Although the concept of a “lipotoxic cardiomyopathy” seems attractive, we propose instead a series of steps on a path from adaptation to maladaptation of the heart in obesity. A case in point is insulin resistance of the heart which may be both adaptive (protecting the heart from excess fuel) or maladaptive (associated with reactive oxygen species formation and activation of signaling pathways of programmed cell death). The present literature reflects an extraordinary complexity of the heart’s metabolic, functional and structural changes in obesity.

Cloning and characterization of a human cDNA ACAD10 mapped to chromosome 12q24.1

Mitochondrial fatty acid beta-oxidation is an important energy resource for many mammal tissues. Acyl-CoA dehydrogenases (ACADs) are a family of flavoproteins that are involved in the beta-oxidation of the fatty acyl-CoA derivatives. Deficiency of these ACADs can cause metabolic disorders including muscle fatigue, hypoglycaemia, hepatic lipidosis and so on. By large scale sequencing, we identified a cDNA sequence of 3960 base pairs with a typical acyl-CoA dehydrogenase function domain. RT-PCR result shows that it is widely expressed in human tissues, especially high in liver, kidney, pancreas and spleen. It is hypothesized that this is a novel member of ACADs family.

NFY interacts with the promoter region of two genes involved in the rat peroxisomal fatty acid β-oxidation: the multifunctional protein type 1 and the 3-ketoacyl-CoA B thiolase

Backgroundβ-oxidation of long and very long chain fatty acyl-CoA derivatives occurs in peroxisomes, which are ubiquitous subcellular organelles of eukaryotic cells. This pathway releases acetyl-CoA as precursor for several key molecules such as cholesterol. Numerous enzymes participating to cholesterol and fatty acids biosynthesis pathways are co-localized in peroxisomes and some of their encoding genes are known as targets of the NFY transcriptional regulator. However, until now no interaction between NFY transcription factor and genes encoding peroxisomal β-oxidation has been reported.ResultsThis work studied the interactions between NFY factor with the rat gene promoters of two enzymes of the fatty acid β-oxidation, MFP-1 (multifunctional protein type 1) and ThB (thiolase B) and their involvement in the cholesterol dependent-gene regulation. Binding of this nuclear factor to the ATTGG motif of the MFP-1 and of the ThB promoters was demonstrated by EMSA (Electrophoretic Mobility Shift Assay) and super shift assay. In contrast, in spite of the presence of putative Sp1 binding sites in these promoters, competitive EMSA did not reveal any binding. The promoter-dependent luciferase gene expression was downregulated by cholesterol in MFP-1 and ThB promoters harbouring constructs.ConclusionsThis work describes for the first time a NFY interaction with promoter sequences of the peroxisomal β-oxidation encoding genes. It suggests that cholesterol would negatively regulate the expression of genes involved in β-oxidation, which generates the initial precursor for its own biosynthesis, via at least the NFY transcription factor.

Properties of the Mouse Intestinal Acyl-CoA:Monoacylglycerol Acyltransferase, MGAT2

Acyl-CoA:monoacylglycerol acyltransferase (MGAT) plays an important role in dietary fat absorption by catalyzing a rate-limiting step in the re-synthesis of diacylglycerols in enterocytes. The present study reports further characterization of MGAT2, a newly identified intestinal MGAT (Cao, J., Lockwood, J., Burn, P., and Shi, Y. (2003) J. Biol. Chem. 278, 13860-13866) for its substrate specificity, requirement for lipid cofactors, optimum pH and Mg2+, and other intrinsic properties. MGAT2 enzyme expressed in COS-7 cells displayed a broad range of substrate specificity toward fatty acyl-CoA derivatives and monoacylglycerols, among which the highest activities were observed with oleoyl-CoA and rac-1-monolauroylglycerol, respectively. MGAT2 appeared to acylate monoacylglycerols containing unsaturated fatty acyls in preference to saturated ones. Lipid cofactors that play roles in signal transduction were shown to modulate MGAT2 activities. In contrast to oleic acid and sphingosine that exhibited inhibitory effects, phosphatidylcholine, phosphatidylserine, and phosphatidic acid stimulated MGAT2 activities. Using recombinant murine MGAT2 expressed in Escherichia coli, we demonstrated conclusively that MGAT2 also possessed an intrinsic acyl-CoA:diacylglycerol acyltransferase (DGAT) activity, which could provide an alternative pathway for triacylglycerol synthesis in the absence of DGAT. In contrast to the inhibitory effect on MGAT2 activities, nonionic and zwitterionic detergents led to a striking activation of DGAT activity of the human DGAT1 expressed in mammalian cells, which further distinguished the behaviors of the two enzymes. The elucidation of properties of MGAT2 will facilitate future development of compounds that inhibit dietary fat absorption as a means to treat obesity.

Kinetics and localization of brain phosphate activated glutaminase.

The cellular concentration of phosphate, the main activator of phosphate activated glutaminase (PAG) is rather constant in brain and kidney. The enzyme activity, however, is modulated by a variety of compounds affecting the binding of phosphate, such as glutamate, calcium, certain long chain fatty acids, fatty acyl CoA derivatives, members of the tricarboxylic acid cycle and protons (Kvamme et al. [2000] Neurochem. Res. 25:1407-1419). Therefore, the kinetic and allosteric properties of the enzyme are essential for regulating the enzyme activity in situ, especially because the enzymically active pool of PAG is assumed to have an external localization in the inner mitochondrial membrane, being exposed to cytosolic variation in the content of effectors. This has largely been overlooked. A hypothetical model for the allosteric interactions based on the sequential induced fit allosteric model by Koshland et al. ([1966] Biochemistry 5:365-385) is presented. Furthermore, it has been generally accepted that there exist only two isoforms of PAG, the kidney PAG that is similar to brain PAG, and the liver PAG. Therefore, the immunoreactivity of brain cells against kidney PAG antibodies has been considered a measure of PAG protein. Gomez-Fabre et al. ([2000] Biochem. J. 345:365-375) recently found, however, that a PAG mRNA from human breast cancer ZR75 cells is present in human brain and liver, but not in the kidney. We observed only traces of PAG immunoreactivity in cultured astrocytes and cultured neuroblastoma cells, regardless whether antibodies against the C- and N-termini of kidney PAG or antibodies against liver PAG were used, but considerable enzyme activity, demonstrating hitherto unknown isoforms of PAG (Torgner et al. [2001] FEBS Lett. 268(Suppl 1):PS2-031).

Effect of exogenous cholesterol and dithiothreitol on the activity of human liver microsomal acyl-coenzyme A:cholesterol acyltransferase (ACAT)

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is the intracellular enzyme responsible for the esterification of cholesterol with long-chain fatty acyl-CoA derivatives and has been implicated in atherosclerosis and gallstone disease. The effects of exogenous cholesterol and dithiothreitol (DTT) on the ACAT activity of human liver microsomes have been determined. Pre-incubation of microsomes with exogenous cholesterol gave a marked stimulation of activity. Experiments with [ 13H]cholesterol and [ 14C]ololeoyl-CoA indicated the time course of equilibration of exogenous with endogenous cholesterol as ACAT substrates, and showed that ACAT activity could be accurately measured using [ 3H]cholesterol/Tween 80, provising that the concentration of endogenous microsomal cholesterol was also determined. Pre-incubation of liver microsomes for 90 min in the presence of 2 mmol/1 DTT and exogenous cholesterol/Tween 80 resulted in a 60% reduction in ACAT activity, compared with the corresponding activity when DTT was omitted. If microsomes were pre-incubated with DTT prior to the pre-incubation with exogenous cholesterol/Tween 80, and 85–90% reduction in ACAT activity occurred. In contrast, pre-incubation of microsomes with DTT in the absence of exogenous cholesterol/Tween 80 (only endogenous cholesterol present) resulted, initially in a stimulation of ACAT activity; on further pre-incubation, activity returned to control levels. These results indicate that the supply of cholesterol to the enzyme active site is an important factor in ACAT assays in vitro and that DTT has a major effect on this process, suggesting that these factors may be important in controlling ACAT activity in vivo.

Interaction of fatty acids, acyl-CoA derivatives and retinoids with microsomal membranes: effect of cytosolic proteins.

This paper reviews characteristics of microsomal membrane structure; long chain fatty acids, acyl CoA derivatives, retinoids and the microsomal formation of acyl CoA derivatives and retinyl esters. It is analyzed how the movement of these molecules at the intracellular level is affected by their respective binding proteins (Fatty acid binding protein, acyl CoA binding protein and cellular retinol binding protein). Studies with model systems using these hydrophobic ligands and the lipid-binding or transfer proteins are also described. This topic is of interest especially because in the esterification of retinol the three substrates and the three binding proteins may interact.

Mechanism of Free Fatty Acid Effects on Hepatocyte Insulin Receptor Binding and Processing

We determined whether the palmitate effects on hepatocyte insulin receptor binding and post-receptor trafficking were mediated by accelerated mitochondrial beta-oxidation or accumulation of intracellular fatty acyl-CoA derivatives and possibly protein acylation. Preincubation of hepatocytes with moderate concentrations of palmitate (0.5 mM) resulted in a 23% decline in cell-surface binding and proportional decreases in receptor-mediated insulin internalization and degradation. Brief pretreatment of hepatocytes with the carnitine palmityltransferase-I inhibitor, methyl palmoxirate (MP), prevented 70% of the palmitate effects. At higher palmitate concentrations (2.0 mM), cell-surface binding was reduced by 34%, whereas internalization of the receptor complex was reduced by 78%. These effects were only partially prevented by MP pretreatment. Receptor-mediated insulin degradation increased by 34% and was uninfluenced by MP pretreatment. Octanoate, which is rapidly shunted into mitochondrial oxidation, produced a dose-dependent reduction in insulin binding, with proportional decreases in internalization and degradation. Similarly preincubation with 2.0 mM oleate, which, unlike palmitate, is not known to produce protein acylation, resulted in proportional decreases in insulin receptor binding and receptor-mediated internalization and degradation. High concentrations of octanoate or oleate (2.0 mM) did not reproduce the additive post-receptor effects of palmitate. We conclude that the receptor and post-receptor effects of moderate palmitate concentrations are closely linked to accelerated fatty acid oxidation. The post-receptor effects observed at higher concentrations involve other mechanisms, possibly relating to intracellular levels of palmityl-CoA derivatives.

Characterization of acetyl-CoA: Ecdysone 3-acetyltransferase in Schistocerca gregaria larvae

Characterization of the acetyltransferase (acetyl-CoA: ecdysone 3-acetyltransferase) which catalyzes the conversion of ecdysone into ecdysone 3-acetate was carried out in gastric caecae of day 7 last instar larvae of Schistocerca gregaria. This enzyme is one of the enzymic systems involved in the inactivation of ecdysteroids. The acetyltransferase exhibited a microsomal subcellular localization, an apparent K m for ecdysone of 71 μM, a maximal specific activity of 7.2 nmol/min/mg of protein and was inhibited competitively in the presence of 20-hydroxyecdysone with K i = 68.8 μM. The enzyme required acetyl-CoA as co-substrate for its activity, the apparent K m for acetyl-CoA being 47.2 μM. Acetic acid could not replace acetyl-CoA as the co-substrate, indicating that the enzyme is an acetyl-CoA: ecdysone acetyltransferase and not a hydrolase. Similarly, esterification of ecdysone was not observed when long-chain fatty acyl-CoA derivatives were substituted as co-substrates. The reaction was linear for 20 min and with protein concentration up to 0.8 mg/ml. The formation of 20-hydroxyecdysone 3-acetate has been demonstrated in the same microsomal fraction and required also acetyl-CoA as co-substrate. The apparent K m of the acetyltransferase for 20-hydroxyecdysone was 53.5 μM, revealing that the enzyme had a somewhat stronger affinity for 20-hydroxyecdysone than for ecdysone.

Purification of lysophosphatidylcholine transacylase from bovine heart muscle microsomes and regulation of activity by lipids and coenzyme A

Heart muscle microsomes catalyze the transacylation of lysophosphatidylcholine (lyso PC) to produce phosphatidylcholine (PC). The enzyme which catalyzes this reaction, lyso PC:lyso PC transacylase, has been isolated and characterized from bovine heart muscle microsomes. The purification of the enzyme was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The purified enzyme was nearly homogeneous and consisted of a single molecular species of 128 kDa as determined by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The catalytic activity of the enzyme was dependent on the presence of either CoA or acyl-CoA, both of which maximally stimulated at concentrations of approx. 10 microM. Analysis of the PC produced in the reaction showed that the enzyme catalyzed a transacylation in which both acyl groups arose from lyso PC. Furthermore, the enzyme did not possess acyl-CoA:lyso PC acyltransferase activity, lysophospholipase or acyl-CoA hydrolase activity, nor did it catalyze transacylation from lyso PC to lysophosphatidylethanolamine, lysophosphatidylinositol or lysophosphatidylserine. Although transacylation was highly specific for lyso PC as the substrate, various unsaturated fatty acyl-CoA derivatives served as activators. Palmitoyl-CoA and stearoyl-CoA did not significantly activate, although acetyl-CoA was an effective activator. Further modulation of activity was produced by palmitic acid and PC, both of which further activated the enzyme in the presence of oleoyl-CoA, whereas arachidonic acid, oleic acid, phosphatidylethanolamine and phosphatidylserine had no effect on activity. The high activity of this transacylase and its regulation by lipids suggests an important role for disaturated PC species in membranes and a mechanism for controlling the metabolism of lyso PC.

Control of cardiac sodium pump by long-chain acyl coenzymes A.

Since we had shown recently that fatty acyl-CoA derivatives stimulate (Na+ + K+)-ATPase activity at suboptimal ATP concentrations, we used sealed vesicles of beef heart sarcolemma to examine the effects of these compounds on the transport function of the enzyme. The sodium pump was detected in inside-out vesicles as a component of Na+ uptake that was dependent on intravesicular (extracellular) K+ and extravesicular (intracellular) ATP and was sensitive to vanadate and digitoxigenin. The pump flux was stimulated without a lag by palmitoyl-CoA (K0.5 = 3 microM) when ATP concentration was 50 microM, but not when it was 2 mM. Saturating palmitoyl-CoA reduced the K0.5 of ATP for the pump by a factor of 3-6. Raising the intracellular K+ concentration increased the K0.5 of ATP, and this effect of K+ was antagonized by palmitoyl-CoA. At concentrations up to 0.5 mM, palmitoyl-CoA had no effect on ATP-independent (passive) Na+ uptake. All tested long-chain acyl-CoA derivatives had effects similar to that of palmitoyl-CoA; but CoA, acetyl-CoA, and palmitic acid were ineffective. Palmitoyl carnitine and docosahexanoic acid, amphiphilic compounds with inhibitory and biphasic effects on the hydrolytic activity of purified (Na+ + K+)-ATPase, had purely inhibitory effects on the pump at high concentrations that also affected the passive fluxes. The data support the proposition that fatty acyl-CoA derivatives mimic the effect of ATP at a regulatory site and suggest that these intracellular liponucleotides may be involved in the control of the pump.

Glutaric aciduria type II: evidence for a defect related to the electron transfer flavoprotein or its dehydrogenase.

Incubation of intact fibroblasts from a patients with glutaric aciduria type II with [2-14C]riboflavin showed normal synthesis of flavin mononucleotide and flavin adenine dinucleotide. This is taken as evidence for normal transport of riboflavin into the cells and normal activity of riboflavin kinase (EC 2.7.1.26) and flavin mononucleotide adenylyltransferase (EC 2.7.7.2). The ability of intact fibroblasts to oxidize 1-14C-fatty acids and [6-14C]lysine is impaired in the patient which together with the urinary excretion pattern of organic acids indicates a defective dehydrogenation of fatty acid acyl-CoAs and glutaryl-CoA. However, dehydrogenation of (C6-C10) fatty acid acyl-CoA derivatives and glutaryl-CoA was normal when the dehydrogenases were measured in fibroblast homogenate with artificial electron acceptors. In vivo, these dehydrogenases transfer their electrons to CoQ10 in the main electron transport chain via electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase. Glutaric aciduria type II fibroblasts showed very diminished activity when the glutaryl-CoA dehydrogenase activity was measured without artificial electron acceptor but with intact endogenous electron transport system. As the NADH and succinate oxidation seems normal in glutaric aciduria type II patients, this is strong evidence for a defect in either the electron transfer flavoprotein or the electron transfer flavoprotein dehydrogenase.

The effect of fatty acid exposure on the biosynthesis of glycerolipids by cultured hepatocytes

Incubation of hepatocyte monolayers with oleate or palmitate (1.0 mM) for 2–48 h, increased (20 to 80%) the incorporation of [1,3- 14C]glycerol and palmitate into triacyglycerol but not phosphatidylcholine. The effect of fatty acids on liver cell triacylglycerol formation correlated well ( r = 0.990) with a simultaneous rise (2–4-fold) in phosphatidate phosphatase (EC 3.1.3.4) activity. Phosphatidate phosphatase activity and triacylglycerol biosynthesis are also increased (2-fold) after hepatocyte monolayers are incubated for 24 h with cyclic GMP in the absence of fatty acids. Fatty acid-dependent increases in liver cell triacylglycerol formation and phosphatidate phosphatase activity are not blocked by cycloheximide. Phosphatidylcholine biosynthesis was also elevated in homogenates of liver cells exposed (24–48 h) to 1.0 mM oleate when exogenous CDPcholine was added to the incubation mixture. Apparently, the phosphatidate phosphatase-dependent rise in diacylglycerols that occurs after fatty acid exposure is primarily shunted into triacylglycerols because liver cell CDPcholine content is not correspondingly increased, and high levels of diacylglycerol acyltransferase (EC 2.3.1.20) and fatty acyl-CoA derivatives are present.

N-acylation of ethanolamine phospholipids in canine myocardium

N-Acylethanolamine phospholipids, which are found in infarcted but not in normal canine myocardium, were produced by preparations of normal myocardial tissue incubated in the presence of millimiolar concentrations of Ca 2+. Biosynthetic activity from endogenous substrates was associated with both microsomal and mitochondrial fractions and exhibited an alkaline pH optimum. The time course of N-acylethanolamine phospholipid synthesis and degradation was followed after pulse labeling of myocardial ethanolamine phospholipids with [1,2- 14C]ethanolamine. Enzymic N-acylation of both phosphatidylethanolamine and lysophosphatidylethanolamine was demonstrated by incubating these substrates with homogenates of myocardial tissue. Neither free fatty acids nor acyl-CoA derivatives served as acyi donor but some of the constituent fatty acids of exogenous phosphatidylethanolamine were recovered among the amide-linked fatty acids of the N-acylethanolainine phospholipids. N-Acylation may thus occur by the transfer of O-acyl groups catalyzed by a Ca 2+-dependent transacylase. Since N-acylethanolamine phospholipids are precursors of the biologically active N-acylethanolamines, their biosynthesis may constitute an important injury-induced metabolic event aimed at the protection of ischwmic myocardial tissue.

Glutamate dehydrogenase activity in developing soybean seed: Isolation and characterization of three forms of the enzyme

Three forms of glutamate dehydrogenase have been isolated from developing soybean seed. Their intracellular locations could not be determined directly because organelles and marker enzymes showed abnormal distribution on sucrose density gradient fractionation. By analogy with enzymes from other parts of the plant, glutamate dehydrogenase 2 was shown to be located in chloroplasts and glutamate dehydrogenase 3 in mitochondria. Glutamate dehydrogenase 1 could not be located in this way because it is found only in the seed. The three enzymes are similar in pH optima, molecular weight and substrate specificity with respect to 2-oxoglutarate and l-glutamate. The mitochondrial enzyme is specific for NAD +. The chloroplast enzyme shows low activity with NADP + relative to NAD + but uses NADPH readily in the aminating direction. Glutamate dehydrogenase 1 is active with both nucleotides and is the only form to show substantial deaminating activity with NADP +. Glutamate dehydrogenases 1 and 2 are activated and stabilized by glutathione and 2-mercaptoethanol whereas enzyme 3 is unaffected. No significant metabolic control of any of the enzymes could be detected. Malate, citrate, adenine nucleotides and long-chain fatty acyl CoA derivatives gave slight inhibition at high concentrations. Amino acids had no effect on activity. A possible role for the enzyme characteristic of the developing seed is discussed in relation to nutrient supply during the accumulation of reserve materials in the seed.

Endogenous formation of prostanoids and the levels of long‐chain fatty acyl CoA derivatives in suspensive and adhesive cells from in vitro embryoid bodies of teratoma OTT 6050

AbstractAttachment of the cell surface to a substratum may play a critical role in initiating some cellular developmental commitments and in sustaining differentiation of cells that have already been specialized. Embryoid bodies of teratoma OTT6050 were divided, on day 10 of initial culture, into myogenic adhesive cells which were already (at day 6) characterized by endogenous prostaglandin (PG)I2 formation and little‐specialized suspensive cells which formed only thromboxane (TX)B2 in the same culture system. Since at day 10 both cell types reached a stationary phase in which the nature of each cell was mature enough for the analyses with mass fragmentographic technique and gas chromatography‐ mass spectrometry (GCMS), the total levels of predominant long‐chain fatty acyl CoA (acyl CoA) derivatives could be measured comparatively as methyl esters after methanolysis. It was found as a result of major differentiation that adhesive cells had a rather low ratio of arachidonyl CoA to stearyl CoA, although adhesive cells accumulated a larger total amount of acyl CoA derivatives than that accumulated in suspensive cells.

Regulation of lipid synthesis in developing adipose tissue

The relationships of hormonal levels and nutritional substrates in the foetus must be considered factors which will affect the later adipose tissue reserve of the infant. De novo lipogenesis has been investigated in developing human adipose tissue at the time of initial lipid accumulation. Measurement of neutral lipid synthesis using 1- 14 C acetate and tritiated water as substrates have shown that de novo fatty acid production proceeds in this tissue and that levels of long chain fatty acyl CoA derivatives and neutral lipid depend on the insulin concentration of the incubation. Insulin activates the lipogenic enzyme, acetyl CoA carboxylase in mature adipose tissue. In developing adipose tissue, in which insulin was demonstrated, acetyl CoA carboxylase activity was measured as 1- 14 C bicarbonate incorporated into malonyl CoA. Enzyme activity increased with gestational age and paralleled both the increasing endogenous triglyceride of the tissue and the in vitro incorporation of acetate to neutral lipid. These increases were shown from the 16th week of gestation. In vitro incorporation studies have shown that increased concentrations of glucose and insulin enhance lipogenesis despite the presence of elevated fatty acid concentrations, which are inhibitory to lipid synthesis in mature tissue.