Search

The maturation of chondrocytes is strictly regulated for proper endochondral bone formation. Although recent studies have revealed that intracellular metabolic processes regulate the proliferation and differentiation of cells, little is known about how changes in metabolite levels regulate chondrocyte maturation. To identify the metabolites which regulate chondrocyte maturation, we performed a metabolome analysis on chondrocytes of Sik3 knockout mice, in which chondrocyte maturation is delayed. Among the metabolites, acetyl-CoA was decreased in this model. Immunohistochemical analysis of the Sik3 knockout chondrocytes indicated that the expression levels of phospho-pyruvate dehydrogenase (phospho-Pdh), an inactivated form of Pdh, which is an enzyme that converts pyruvate to acetyl-CoA, and of Pdh kinase 4 (Pdk4), which phosphorylates Pdh, were increased. Inhibition of Pdh by treatment with CPI613 delayed chondrocyte maturation in metatarsal primordial cartilage in organ culture. These results collectively suggest that decreasing the acetyl-CoA level is a cause and not result of the delayed chondrocyte maturation. Sik3 appears to increase the acetyl-CoA level by decreasing the expression level of Pdk4. Blocking ATP synthesis in the TCA cycle by treatment with rotenone also delayed chondrocyte maturation in metatarsal primordial cartilage in organ culture, suggesting the possibility that depriving acetyl-CoA as a substrate for the TCA cycle is responsible for the delayed maturation. Our finding of acetyl-CoA as a regulator of chondrocyte maturation could contribute to understanding the regulatory mechanisms controlling endochondral bone formation by metabolites.

Antizyme (AZ) regulates cellular polyamines (i.e., putrescine, spermidine, and spermine) through binding to ornithine decarboxylase and subsequent ubiquitin-independent degradation of the enzyme protein by the 26S proteasome. Screening for AZ-binding proteins using a yeast two-hybrid system identified ATP citrate lyase (ACLY), a cytosolic enzyme which catalyzes the production of acetyl-CoA that is used for lipid anabolism or acetylation of cellular components. We confirmed that both AZ1 and AZ2 bind to ACLY and AZ colocalizes with ACLY to the cytoplasm. Unexpectedly, neither AZ1 nor AZ2 accelerated ACLY degradation. Additionally, purified AZ, particularly AZ1, increased the activity of purified ACLY in a dose-dependent manner in vitro, suggesting that AZ activates ACLY through protein-protein interaction. Polyamines themselves had no effect on the ACLY activity in vitro. Knockdown of AZ1 and/or AZ2 in human cancer cells significantly decreased the ACLY activity as well as cellular levels of acetyl-CoA and cholesterol. Our results are the first to show the crosstalk between polyamine and acetyl-CoA metabolism. We hypothesize that AZ may promote acetyl-CoA synthesis to downregulate spermidine and spermine through acetylation.

It is known that biotin prevents the development of diabetes by increasing the functions of pancreatic beta-cells and improving insulin sensitivity in the periphery. However, its anti-obesity effects such as anorectic effects remain to be clarified. Acetyl CoA carboxylase (ACC), a biotin-dependent enzyme, has two isoforms (ACC1 and ACC2) and serves to catalyze the reaction of acetyl CoA to malonyl CoA. In the hypothalamus, ACC2 increases the production of malonyl CoA, which acts as a satiety signal. In this study, we investigated whether biotin increases the gene expression of ACC2 in the hypothalamus and suppresses food intake in mice administered excessive biotin. Food intake was significantly decreased by biotin, but plasma regulators of appetite, including glucose, ghrelin, and leptin, were not affected. On the other hand, biotin notably accumulated in the hypothalamus and enhanced ACC2 gene expression there, but it did not change the gene expression of ACC1, malonyl CoA decarboxylase (a malonyl CoA-degrading enzyme), and AMP-activated protein kinase α-2 (an ACC-inhibitory enzyme). These findings strongly suggest that biotin potentiates the suppression of appetite by upregulating ACC2 gene expression in the hypothalamus. This effect of biotin may contribute to the prevention of diabetes by biotin treatment.

Three coenzyme A (CoA) molecular species, i.e., acetyl-CoA, malonyl-CoA, and nonesterified CoA (CoASH), in 13 types of fasted rat tissue were analyzed. A relatively larger pool size of total CoA, consisting of acetyl-CoA, malonyl-CoA, and CoASH, was observed in the medulla oblongata, liver, heart, and brown adipose tissue. Focusing on changes in the CoA pool size in response to the nutrient composition of the diet given, total CoA pools in rats continuously fed a high-fat diet for 4 weeks were significantly higher in the hypothalamus, cerebellum, and kidney, and significantly lower in the liver and skeletal muscle than those of rats fed a high-carbohydrate or high-protein diet. In particular, reductions in the liver were remarkable and were caused by decreased CoASH levels. Consequently, the total CoA pool size was reduced by approximately one-fifth of the hepatic contents of rats fed the other diets. In the hypothalamus, which monitors energy balance, all three CoA molecular species measured were at higher levels when rats were fed the high-fat diet. Thus, it was of interest that feeding rats a high-fat diet affected the behaviors of CoA pools in the hypothalamus, liver, and skeletal muscle, suggesting a significant relationship between CoA pools, especially malonyl-CoA and/or CoASH pools, and lipid metabolism in vivo.

Levels of three coenzyme A (CoA) molecular species, i.e., nonesterified CoA (CoASH), acetyl-CoA, and malonyl-CoA, in fasted and fed rat tissues were analyzed by the acyl-CoA cycling method which makes detection possible at the pmol level. Malonyl-CoA in brain tissues readily increased with feeding, and inversely, acetyl-CoA decreased. This phenomenon occurred in the cerebral cortex, hippocampus, cerebellum, and medulla oblongata, as well as in the hypothalamus which controls energy balance by monitoring malonyl-CoA. In the non-brain tissues, the sizes of the acetyl-CoA, malonyl-CoA, and CoASH pools depended on the tissues. The total CoA pools consisting of the above three CoA species in the liver, heart, and brown adipose tissue were larger and those of the perirenal, epididymal, and ovarian adipose tissues were much smaller, compared with those of other tissues including brain tissues. In addition, the response of each CoA pool to feeding was not uniform, suggesting that the tissue-specific metabolism individually functions in the non-brain tissues. Thus, a comprehensive analysis of thirteen types of rat tissue revealed that CoA pools have different sizes and showed a different response to fasting and feeding depending on the tissue.

2-Amino-3-ketobutyrate can be readily formed enzymatically by the action of L-threonine dehydrogenase. A convenient assay for determining the half-life of this beta-keto acid is afforded by its rapid and quantitative conversion to glycine (+ acetyl CoA), as catalyzed by 2-amino-3-ketobutyrate CoA lyase. Using this system, we have found the half-life of 2-amino-3-ketobutyrate varies with pH from 8.6 minutes at pH 5.9 to 140 minutes at pH 11.1 yielding a theoretical titration curve that predicts a pKa value of 8.15 for the alpha-amino group of this intermediate. These data are considered relevant to discussions pertaining to a threonine dehydrogenase/2-amino-3-ketobutyrate CoA lyase enzyme complex in the threonine utilization pathway and to mechanistic aspects of the 5-aminolevulinate synthase-catalyzed reaction where 2-amino-3-ketoadipate is involved.

Summary Acetyl CoA: deacetylcephalosporin C o-acetyltransferase(DCPC-ATF) catalyses the final step in the biosynthesis of cephalosporin C ( CPC ) in Acremonium chrysogenum . The gene encoding DCPC-ATF, cefG, has been isolated from an A . chrysogenum genomic library using a DCPC-ATF cDNA probe. Nucleotide sequence analysis revealed that cefG contains two short introns of 79bp and 65bp. The gene was found to be closely linked to the cefEF gene encoding deacetoxycephalosporin C synthetase / deacetylcephalosporin C synthetase, which catalyses the preceding two steps in the pathway. The two genes are separated by a 1114 by segment from which they are divergently transcribed. Introduction of the cloned cefG gene to A.chrysogenum resulted in an increased level of DCPC-ATF activity. A plasmid carrying a cefG gene interrupted in the coding region by a selectable marker for resistance to hygromycin B was constructed and used to disrupt the cefG locus in A.chrysogenum . The cefG-disrupted strains were found to lack the ability to produce CPC, and accumulated its precursor, deacetylcephalosporin C in the culture broth. Southern hybridization analysis confirmed that the disruption resulted from a gene replacement event at the cefG locus.

To determine the role of hydrophobic interactions in the dimer interface of citrate synthase (CS) from Thermoplasma (Tp) acidophilum in thermostabilization, we have used site-directed mutagenesis to replace Gly 196 by Val on the helix L of the subunit interface. Recombinant wild-type and Gly 196 mutant TpCS enzymes were largely identical in terms of substrate specificities (Km for oxaloacetate and acetyl CoA). However, the mutation not only reduced catalytic activity (about 10-fold) (i.e., Vmax, kcat and specific activity) of the TpCS, but also decreased its thermal and chemical stability. Archaeal citrate synthase is active as a dimer, since residues from both monomers participate in the active site. Our results suggest that Gly196 → Val mutation interferes with dimerization, so that improper dimerization or dissociation of the dimer would have a profound affect on the activity as well as the conformational stability of TpCS.

Acetyl CoA: deacetylcephalosporin C o-acetyltransferase(DCPC-ATF) catalyses the final step in the biosynthesis of cephalosporin C, the conversion of deacetylcephalosporin C to cephalosporin C. A cDNA encoding DCPC-ATF has been isolated from a cDNA library of a cephalosporin C producing fungus Acremonium chrysogenum using oligonucleotide probes based on N-terminal amino acid sequences of the enzyme. The cDNA contains a single large open reading frame for a putative precursor consisting of 12 amino acid(AA) leader peptide of unknown function, 274 AA large subunit and 126 AA small subunit at the carboxyl end. The cDNA was expressed in yeast exhibiting a functional DCPC-ATF activity. It was also indicated that the leader peptide was not essential for expression of the enzyme activity. The primary structure of DCPC-ATF shows significant homology with those of acetyl CoA: homoserine o-acetyltransferase in Saccharomyces cerevisiae and Ascobolas immersus.

The synaptic membrane (SM) prepared from rat brain synaptosomes displayed the highest activity of phospholipase D of mammalian tissue thus far observed. In common with the previously described latency of the microsomal enzyme, the SM phospholipase D (PLD) activity is barely detectable in the absence of 4 mM sodium oleate. Incubation of synaptosomes in the presence of choline labelled lecithin, sodium oleate and acetyl CoA resulted in the formation of a product co - chromatographing with acetylcholine. The potential role of SM PLD liberating choline from lecithin into the synaptic cleft is discussed in relationship to acetylcholine formation.

In isolated rat heart mitochondria incubated with excess ADP and malate, α-ketoisocaproate (KIC) at 0.05–0.1 mM more than doubled the rate of citrate cycle activity (measured as citrate accumulation in the presence of fluorocitrate). Replacement of fluorocitrate with arsenite increased pyruvate formation stoichiometrically with the previous increase in citrate formation. No enhancement of pyruvate or citrate accumulation was seen when malate was omitted or when KIC was replaced with equimolar leucine. KIC (leucine) did not give rise to acetyl CoA, nor was pyruvate formed by way of oxaloacetate and phosphoenolpyruvate. These findings suggest that KIC regulated the formation of pyruvate from malate by acting as a positive modulator of mitochondrial malic enzyme.

Summary The relative contributions of lactate, propionate and octanoate to the acetyl CoA pool for ketone body formation in the perfused guinea pig liver was investigated with 14 C-labeled substrates. In the presence of octanoate, incorporation of 3-( 14 C)-lactate or 3-( 14 C)-propionate into acetoacetate was extremely low, amounting to only about 0.2% of the incorporation of 1-( 14 C)-octanoate which accounted for 28% of the acetoacetate synthesized, when this labeled fatty acid was perfused together with un-labeled lactate or propionate. Incorporation of 3-( 14 C)-lactate and 3-( 14 C)-propionate into CO 2 was likewise very low, in the same experiments amounting only to less than 0.7% and 7.0% respectively of the 1-( 14 C)-octanoate carbon oxidized to CO 2 in the presence of unlabeled lactate or propionate. Despite the potential availability of acetyl CoA from lactate via pyruvate; and from propionate via succinate, oxalacetate, P-enolpyruvate and pyruvate, its formation was minimal. The results indicate that pyruvate dehydrogenase is strongly inhibited during ketogenesis, when under these conditions, there is virtually complete conversion of metabolic pyruvate to glucose.

Evidence is presented for the thiol reagent methyl methanethiolsulfonate inhibiting choline acetyltransferase (EC 2.3.1.6), not by reaction with an enzymic thiol group, but by reaction with the thiol group of CoA. The resulting CoA methyl disulfide is a potent inhibitor of this enzyme. Its action is reversed competitively by acetyl CoA.

Extramitochondrial malic enzyme (EC 1.1.1.40) of rat liver and skeletal muscle is shown for the first time to be subject to significant inhibition by acetyl CoA. The liver enzyme seems to be slightly more sensitive to inhibition than the muscle enzyme, and inhibition can only be observed at malate concentrations below Vmax. Since acetyl CoA is a metabolite of ethanol (acetate) oxidation in liver and muscle, its inhibitory effect on malic enzyme could explain (a) the prolonged disequilibrium of the liver malic enzyme reaction after acute ethanol injection in rats (1), and (b) the inhibition of alanine release from perfused rat hindquarters in the presence of acetate, the major metabolite of ethanol in the peripheral vascular system (2).

Rat liver nuclei were incubated for various lengths of time in the presence of increasing concentrations of acetyl CoA. The rate of acetylation varied strongly according to the acetyl CoA concentration. A very small part of the histone was acetylated. After incubation in the presence of increasing acetyl CoA concentrations, four apparent Km could be determined. Electrophoretic analysis showed that only histone H3 was acetylated which suggests that each Km corresponds to a sequential acetylation of its lysine residues. This could be correlated with the possible role of histone acetylation in the control of gene activity.

Summary The activities of hydroxymethylglutaryl (HMG) CoA synthetase and lyase, acetoacetyl CoA thiolase and deacylase were measured in mitochondria of human adipose tissue. All enzymes with the exception of HMG CoA synthetase, were found to be very active. Our results strongly suggest that acetoacetate can be formed in these mitochondria due to the presence of an active acetoacetyl CoA deacylase and that in this tissue, where the activity of the citrate cleavage enzyme is very low, acetyl CoA units leave mitochondria in the form of acetoacetate to participate in cytoplasmic fatty acid synthesis.

The activity of ATP citrate lyase, the enzyme responsible for production of cytoplasmic acetyl CoA for lipogenesis, is known to be modulated by dietary manipulation. The present work suggests a mechanism for control of the rate of its degradation. An unusual peptide factor, shown earlier by Dunaway and Segal [J. Biol. Chem. 251, 2323–2329 (1976)] to stabilize phosphofructokinase from thermal or lysosomal degradation, exerts similar stabilizing effects on ATP citrate lyase. These experiments confirm that the effects of the stabilizing factor are pleitropic, and suggest that it may play a role in the control of turnover of other lipogenic enzymes whose activities are similarly modulated.

We have reported that rats given 10–20 mmol/kg of propionate develop hyperammomemia in response to amino acid loads. Ethanol in doses of 0.1–5 mmoles/kg attenuates or prevents this hyperammonemia. Liver ATP, glutamate and aspartate levels are unaffected, but the fall in acetyl CoA and secondarily in N-acetylglutamate caused by propionate alone is much reduced. As a result of this effect, mitochondrial carbamoyl phosphate synthetase activity is restored nearly to normal. Thus low-dose ethanol combats this form of hyperammonemia by augmenting hepatic acetyl CoA.

Acute and chronic treatment with clofibrate increased the total CoA content of rat liver and altered the profile of the various CoA thioesters. There resulted a 2–3 fold increase in the contents of long chain acyl CoA, acetyl CoA and free CoA, contrasting with significant decreases found in succinyl CoA, malonyl CoA and acetoacetyl CoA contents. It is postulated that the known increase in fatty acid binding protein and/or the increased extramitochondrial β-oxidation of fat by an increased peroxisomal population may direct the compartmentation and metabolic fate of fatty acids and their CoA derivatives following clofibrate treatment.

Summary A novel type of pyruvate oxidase, which catalyzes acetylation of CoA in the presence of pyruvate, oxygen and FAD, was detected in Entamoeba histolytica . The enzyme was partially purified by centrifugation, ammonium sulfate precipitation and hydroxyapatite chromatography. Enzymatic activity was found in two fractions. The ratio of oxygen consumed to acetyl CoA produced was 1 with the purified enzyme(s). In the presence of excess catalase the velocity of oxygen uptake was one-half. Therefore, the postulated reaction is: pyruvate + CoA + 02 = acetyl CoA + CO2 + H202. For this new enzyme, we propose the trivial name of pyruvate oxidase (CoA acetylating).

The carboxylation of pyruvate by human adipose tissue mitochondria was found to be very low as compared to that by rat adipose tissue mitochondria. This finding supports the previous reports of neglegible fatty acid synthesis from glucose by human adipose tissue and underlines an important role of this process in lipogenesis. It is suggested that the availability of intramitochondrial oxaloacetate limits a translocation of acetyl CoA as citrate from the mitochondria to the cytosol. In contrast to an age-dependency of mitochondrial metabolism in rat adipose tissue, pyruvate carboxylation by human adipose tissue mitochondria does not respond to aging.

Citrate cleavage enzyme has been detected in mango fruit. The enzyme activity increases considerably during ripening of the fruit and is stimulated by glucose, fructose and fatty acids (oleic and palmitic). This enzyme appears to be of major importance in the ripening fruit in making available acetyl CoA and oxaloacetate, from citrate, for synthetic processes.

Glucagon has recently been shown to have a direct lipolytic action on liver (Bewsher and Ashmore 1966). Increased hepatic lipolysis, plus enhanced availability of plasma fatty acids, causes an elevation of acetyl CoA and fatty acyl CoA levels (Williamson, Herczeg, Coles and Danish 1966). Such elevated levels of acetyl CoA presumably account for the increased rate of ketogenesis observed with glucagon. Acute effects of insulin deficiency, induced by administration of guinea pig anti-insulin serum (AIS) to rats, are similar to effects produced by glucagon: elevation of plasma glucose, free fatty acid, and ketone levels, plus an increased rate of gluconeogenesis (Wagle and Ashmore, 1963 ; Wagle and Ashmore, 1964 ; Tarrant, Mahler, and Ashmore, 1964) . These findings have led to the suggestion that the early metabolic changes in experimental insulin deficiency may be induced by hormones with actions normally counterbalanced by insulin (Wright, 1965) . Such hormones are glucagon, epinephrine, ACTH and corticosteroids (Mahler, Stafford, Tarrant and Ashmore, 1964 ; Jungas and Ball, 1963) . Data presented in this paper supports the concept that the lipolytic, ketogenic, and gluconeogenic effects of glucagon are revealed in the insulin-deficient rat. It is suggested that stimulation of gluconeogenesis, both with glucagon and with AIS, is secondary to enhanced lipolysis and is mediated through facilitation of pyruvic carboxylase by elevated hepatic levels of acetyl CoA.

The isolation of β-carboxy, β-hydroxyisocaproic acid, its characterization, and its enzymatic synthesis from α-ketoisovalerate and acetyl COA was described in a previous communication (Jungwirth et al , 1961) . This note reports the isolation of another compound from culture filtrates of a mutant of Neurospora crassa , its enzymatic conversion to α-ketoisocaproic acid, and leucine by extracts of Salmonella typhimurium and Neurospora . The compound has been tentatively identified as α-hydroxy, β-carboxyisocaproic acid.

Initial velocity studies of the reaction catalyzed by pyruvate carboxylase from rat liver have defined the properties of oxalacetate synthesis in the absence of acetyl-CoA. The maximal rate of oxalacetate synthesis under these conditions is 21% of that observed in the presence of saturating concentrations of this activator. The studies have also indicated that marked changes are observed in the apparent KM (HCO3−), or velocity/concentration profile (MgATP2−), for the substrates of the carboxylation partial reaction as a function of [acetyl-CoA] over the range 0 – 10μM with little change in the apparent VMAX.

Reaction of α-isopropylmalate synthase from Salmonella typhimurium with the bifunctional cross-linking reagent dimethyl suberimidate and subsequent sodium dodecyl sulfate gel electrophoresis results in the appearance of four protein species with molecular weights of about 50,000, 100,000, 150,000, and 200,000. In the absence of ligands, the pattern consists predominantly of tetramers and dimers (“native” pattern). This pattern is changed to one of dimers and monomers in the presence of inhibitory concentrations of leucine. Acetyl-CoA, known to kinetically compete with leucine, restores the “native” pattern. On the basis of previous findings, the results are discussed in terms of “frozen” equilibria.

The entire sequence of enzymes of the pentose phosphate pathway, the glycolytic pathway and certain enzymes of fatty acid synthesis were measured in developing rat brain. Coordinated increases in enzymes of the glycolytic pathway were observed, with apparent discrimination in that greater changes were found among enzymes of low turnover capability (Vmax: Km). During development a sequential pattern emerged with early changes in phosphofructokinase and aldolase followed by triosephosphate isomerase, glycerophosphate dehydrogenase and phosphoglyceromutase; enzymes of the pentose phosphate pathway remained relatively constant, while a marked and parallel fall was seen in acetyl CoA car☐ylase, fatty acid synthetase and NADP+-linked isocitrate dehydrogenase.

An enzyme has been partially purified from Klebsiella aerogenes which transfers an acetyl group from S-acetyl phosphopantetheine to deacetyl citrate lyase. This converts the deacetyl citrate lyase which has no enzyme activity, to citrate lyase, the active enzyme. A variety of other acetyl thioesters including acetyl CoA did not serve as acetyl donors.

The active-site-directed reagent, bromopyruvate has been used to covalently label the pyruvate binding site of pyruvate carboxylase (E.C.6.4.1.1.) isolated from sheep liver. Oxalo-acetate proved to be the most effective reaction component in protecting the enzyme against inactivation; pyruvate was less effective although its efficiency was enhanced by the presence of acetyl CoA. The other reaction components, MgATP2− and HCO3− failed to protect the enzyme against inactivation. Using bromo[214C]pyruvate, it was shown that at 100% inactivation, 1.5 pyruvyl residues were bound per mole of biotin and when the reaction was carried out in the presence of acetyl CoA, this ratio was reduced to 1.0. Analysis of pronase digests of the enzyme revealed that more than 90% of the radioactivity was present as carboxy-hydroxyethyl cysteine.

Summary Buffer-soluble extracts of acetate-grown Methanosarcina barkeri catalyzed methanogenesis from acetate in the presence of hydrogen and ATP. The rates of methane formation from either acetate plus ATP, or acetylphosphate without ATP added, were approximately doubled by the addition of coenzyme A (CoA). In vitro methyl group transfer from [2-14C]acetate to form [14CH3]methyl coenzyme M (14CH3S-CoM) was monitored by causing the accumulation of 14CH3S-CoM (14CH3-SCH2CH2SO3−) in the presence of 2-bromoethanesulfonate. The rate of 14CH3S-CoM formation was increased 2.5-fold by 0.2 mM CoA.

In hepatocytes isolated from fed rats, low concentrations of oxalate (50 to 100 μM) greatly enhance ketogenesis and decrease fatty acid synthesis. These metabolic changes are due to pyruvate carboxylase inhibition. Dichloroacetate, which can be catabolized into oxalate enhances ketogenesis only when cells are enriched with lactate and pyruvate and has no obvious effect on lipogenesis. The enhancement of ketogenesis, in both cases, is due to an imbalance between pyruvate dehydrogenase and pyruvate carboxylase but with oxalate, the primary event is oxaloacetate shortage and with dichloroacetate, mitochondrial acetyl CoA excess. This work demonstrates that the studied effects of dichloroacetate are not mediated by oxalate and that low concentrations of oxalate alter the lipid metabolism of hepatocytes.

The effects of secretin on polyamine metabolism in rat pancreas were investigated. Single injections of secretin increased ornithine decarboxylase activity only very slightly. However a substantial time- and dose-dependent increase of acetyl CoA: polyamine N 1 -acetyltransferase activity was observed. The concentrations of N 1 -acetylspermidine, putrescine and β-alanine increased concomitantly, but spermidine and spermine remained unchanged. These results suggest that, in this model, the accumulated putrescine was formed from spermidine, via its acetylation, rather than from ornithine.

Streptomyces alboniger produces the antibiotic puromycin and expresses an enzymic activity which acetylates the drug using acetyl CoA. The N-acetyl-puromycin formed is biologically inactive against protein synthesis in Bacillus subtilis (as assayed in vivo).

Summary Measurements of citrate formation and endogenous adenine nucleotides in fluorocitrate-inhibited rat liver mitochondria respiring in the presence of 50 μM (−) palmitoyl carnitine and 250 μM malate have failed to support the suggestion that citrate synthase activity is controlled primarily by the intramitochondrial ATP level. A positive correlation was obtained between the redox state of the mitochondrial pyridine nucleotides and the rate of citrate formation, suggesting that at physiological malate concentrations, the rate of entry of acetyl CoA into the citric acid cycle is effectively controlled by oxalacetate availability. As acetyl CoA levels increase, and oxalacetate levels decrease, in rat liver with increased fatty acid availability (Williamson, Herczeg, Coles and Danish, 1966 ; Williamson, 1967) , increased ketogenesis accompanies the observed increase in the redox state of pyridine nucleotides in both the cytoplasmic and mitochondrial spaces. We suggest that the change in the redox state following increased fatty acid oxidation is a primary event preceding ketosis.

Summary Citrate synthase from mango fruit differs from other aerobic citrate synthases in that it is inhibited by several sulfhydryl binding reagents. The enzyme is protected from inhibition by its substrates acetyl CoA and oxaloacetate. This enzyme is an “S-citrate synthase” with a molecular weight of 65,000.

Glucagon administered by intravenous infusion to rats in viv has been shown to stimulate ketogenesis and increase levels of acetyl CoA and fatty acyl CoA in the liver. This effect was observed in fed rats, where it was prompt and transient, but not in rats unfed for 24 hours. These results substantiate that glucagon has a lipolytic effect in liver. The ketogenesis induced either by glucagon or starvation was associated with elevated levels of citrate and decreased levels of oxalacetate. A relationship between acetyl CoA and acetoacetate levels in the liver is described.

Mitochondria obtained from mammalian liver are capable of incorporating acetyl CoA into long chain fatty acids. Some of the characteristics of synthesis of fatty acids within mitochondria have been reported (1,2). The purpose of this communication is to present evidence for the existence of several pathways of synthesis within mitochondria and to indicate the important role of mitochondria in the synthesis and alteration of unsaturated fatty acids.

Sheep kidney pyruvate carboxylase has been desensitized against its allosteric effector, acetyl CoA, by limited covalent modification with trinitrobenzene sulphonic acid. Trinitrophenylation of the enzyme resulted in a strong inhibition of the rate of the acetyl CoA-stimulated pyruvate carboxylation and enhancement of the rate of the acetyl CoA-independent reaction. A good correlation was found between the requirement for acetyl CoA of the exchange reactions catalysed by the enzyme and the extent of their inhibition by trinitrobenzene sulphonic acid modification. Spectrophotometric data indicated that one to two lysyl residues per monomer were trinitrophenylated. Modification had only a slight effect on the sedimentation properties of the enzyme.

A cell-free preparation of the yeast, Hansenula ciferri catalyzed the transfer of the acetyl group of acetyl coenzyme-A to the sphinosine bases at both their amino and hydroxyl groups. The enzyme also transferred acetyl groups to the hydroxyls of the N-acetylated sphingosine bases as well as to the amino groups of primary amines of ten or more carbon atoms. A mixture of acetate, ATP and coenzyme-A could be employed instead of acetyl CoA. The reaction had an optimal pH at about 7.8 and was inhibited by free coenzyme-A.

The soluble fatty acid synthetase from E. coli produces both saturated and unsaturated long-chain fatty acids from malonyl CoA and acetyl CoA in a process dependent on TPNH and ACP ( Lennarz, Light and Bloch, 1962 ; Goldman and Vagelos, 1962 ). We have now observed that under certain conditions the synthesis of long-chain acids is suppressed and almost entirely replaced by the production of triacetic acid (TAA, 3, 5-diketohexanoic acid). This communication describes the isolation and characterization of TAA as its lactone (TAL), and details the conditions under which it is formed at the expense of long-chain acids.