Determination of pyruvate dehydrogenase and acetyl-CoA synthetase activities using citrate synthase

Methods are described for the assay of pyruvate dehydrogenase and acetyl-CoA synthetase activities in rat brain subcellular fractions. Citrate synthase and oxaloacetate serve as a trapping system in these assays. The methods permit the determination of a large number of samples of different turbidity with satisfactory precision. Highest activities of pyruvate dehydrogenase and acetyl-CoA synthetase (117.7 and 7.29 nmol/min/mg of protein, respectively) were found in rat brain mitochondria. A three times lower activity of acetyl-CoA synthetase and negligible of pyruvate dehydrogenase was found in brain cytosol.     

Enzyme systems of the substrate and cofactor supply of hyperlipogenesis in non-insulin-dependent diabetes

Enzymatic systems of hepatic hyperlipogenesis supply by substrate (acetyl-CoA) and cofactors (NADPH and ATP) were studied in experiments on diabetic C57Bl/Ks J mice (db/db) that served as a model of non-insulin dependent diabetes. The rise in acetyl-CoA synthetase activity catalyzing the primary step of lipogenesis from acetate has been found, while pyruvate dehydrogenase complex activity did not differ from the control and ATP-citrate lyase activity was lowered. Hyperlipogenesis in non-insulin dependent diabetes was induced by the activation of cellular energy supply revealed in enhanced 2-oxoglutarate dehydrogenase activity and elevated ATP level, as well as changes in the activity ratio of NADPH supply and utilization and the rise in fructose-1,6-diphosphate, allosteric effector of fatty acid synthetase, which resulted in the increase of the enzyme activity and created wider potentials of NADPH utilization as a reducing equivalent in lipogenesis.     

Acetyl-CoA Synthesizing Enzymes in Cholinergic Nerve Terminals

The activities of five enzymes involved in acetyl-CoA synthesis, pyruvate dehydrogenase complex, ATP citrate lyase, carnitine acetyltransferase, acetyl-CoA synthetase, and citrate synthase, were determined in normal nucleus interpeduncularis and nucleus interpeduncularis in which cholinergic terminals were removed following lesion of the habenulointerpeduncular tract. The activities of aspartate transaminase, fumarase, and GABA transaminase also were determined to compare the effect of lesion on other mitochondrial enzymes which are not linked to the biosynthesis of ACh. In normal nucleus interpeduncularis the activities of carnitine acetyltransferase and pyruvate dehydrogenase complex were higher than the activity of ChAT (choline acetyltransferase), whereas the activities of acetyl-CoA synthetase and citrate synthase were considerably lower than that of ChAT. The effect of the lesion separated the enzymes into two groups: the activities of pyruvate dehydrogenase complex, carnitine acetyltransferase, fumarase and aspartate transaminase decreased by 30--40%, whereas the activities of the other enzymes descreased 5--15%. ChAT activity was in all cases less than 15% of normal. It could be concluded that none of the acetyl-CoA synthesizing enzymes decreased to the degree that ChAT did. Only pyruvate dehydrogenase complex and carnitine acetyltransferase seem to be localized in cholinergic terminals to a significant degree. ATP citrate lyase as well as acetyl-CoA synthetase seem to have less significance in supporting acetyl-CoA formation in cholinergic nerve terminals.     

Occurrence and metabolic role of the pyruvate dehydrogenase complex in the lower termite Coptotermes formosanus (Shiraki)

The activities of the pyruvate dehydrogenase complex in extracts of the gutted body, head, foregut/midgut and hindgut (hindgut epithelium and microorganisms) tissues of the lower termite Coptotermes formosanus (Shiraki) were determined by measuring the [¹⁴C]-acetyl-CoA produced from [2-¹⁴C]-pyruvate and the ¹⁴CO₂ produced from [1-¹⁴C]-pyruvate. The activities of pyruvate dehydrogenase, l-lactate dehydrogenase, acetyl-CoA synthetase, malate dehydrogenase (decarboxylating), and acetate kinase in the termite tissues and the hindgut also were determined. The sum (7.1 nmol/termite/h) of the pyruvate dehydrogenase complex activities in the termite tissues other than the hindgut was five times higher than the activity in the hindgut. Significant amounts of l-lactate dehydrogenase activity were found in all of the tissues. All of the tissues other than the hindgut had significant amounts of acetyl-CoA synthetase activity. Malate dehydrogenase (decarboxylating) activity was about ten times higher in the hindgut extract than in the gutted body and head extracts and the activity in the foregut/midgut extract was very low. These results indicate that acetyl-CoA for the TCA cycle is produced effectively in the tissues of the termite itself, both from pyruvate by the pyruvate dehydrogenase complex and from acetate by acetyl-CoA synthetase.     

Interactions between alpha-ketoisovalerate metabolism and the pathways of gluconeogenesis and urea synthesis in isolated hepatocytes

The mechanism of inhibition of pyruvate carboxylase, pyruvate dehydrogenase, and carbamyl phosphate synthetase induced by alpha-ketoisovalerate metabolism has been investigated in isolated rat hepatocytes incubated with lactate, pyruvate, ammonia, and ornithine as substrates. Half-maximum inhibitions of flux through each of these enzyme steps were obtained with 0.3 mM alpha-ketoisovalerate. The inhibition of pyruvate carboxylase flux by alpha-ketoisovalerate was largely reversed by oleate addition, but pyruvate dehydrogenase flux was inhibited further. Inhibition of flux through pyruvate carboxylase could be attributed mainly to the fall of its allosteric activator, acetyl-CoA, with some additional effect due to inhibition by methylmalonyl-CoA. Tissue acetyl-CoA levels decrease as a result of an inhibition of the active form of pyruvate dehydrogenase. Kinetic studies with the purified pig heart pyruvate dehydrogenase complex showed that methyl-malonyl-CoA, propionyl-CoA, and isobutyryl-CoA were inhibitory, the latter noncompetitive with CoASH with an apparent Ki of 90 microM. The observed inhibition of pyruvate dehydrogenase flux correlated with increases of the acetyl-CoA/CoASH and propionyl-CoA/CoASH ratios and isobutyryl-CoA levels, while increases of the mitochondrial NADH/NAD+ ratio explained differences between the effects of alpha-ketoisovalerate and propionate. Carbamyl phosphate synthetase I purified from rat liver was shown to be inhibited directly by methylmalonyl-CoA (apparent Ki of 5 mM). Inhibition of flux through carbamyl phosphate synthetase during alpha-ketoisovalerate metabolism could be attributed both to a direct inhibitory effect of methyl-malonyl-CoA and to a diminished activation by N-acetylglutamate. Direct effects of various acyl-CoA metabolites on these key enzymes may explain symptoms of hypoglycemia and hyperammonemia observed in patients with inherited disorders of organic acid metabolism.     

The activities of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase in hearts and mammary glands from ruminants and non-ruminants.

1. The activities of 2-oxoglutarate dehydrogenase (EC 1.2.4.2) were measured in hearts and mammary glands of rats, mice, rabbits, guinea pigs, cows, sheep, goats and in the flight muscles of several Hymenoptera. 2. The activity of 2-oxoglutarate dehydrogenase was similar to the maximum flux through the tricarboxylic acid cycle in vivo. Therefore measuring the activity of this enzyme may provide a simple method for estimating the maximum flux through the cycle for comparative investigations. 3. The activities of pyruvate dehydrogenase (EC 1.2.4.1) in mammalian hearts were similar to those of 2-oxoglutarate dehydrogenase, suggesting that in these tissues the tricarboxylic acid cycle can be supplied (under some conditions) by acetyl-CoA derived from pyruvate alone. 4. In the lactating mammary glands of the rat and mouse, the activities of pyruvate dehydrogenase exceeded those of 2-oxoglutarate dehydrogenase, reflecting a flux of pyruvate to acetyl-CoA for fatty acid synthesis in addition to that of oxidation via the tricarboxylic acid cycle. In ruminant mammary glands the activities of pyruvate dehydrogenase were similar to those of 2-oxoglutarate dehydrogenase, reflecting the absence of a significant flux of pyruvate to fatty acids in these tissues.     

Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.     

Molecular biology of acetyl-CoA metabolism

We have characterized the expression of potential acetyl-CoA-generating genes (acetyl-CoA synthetase, pyruvate decarboxylase, acetaldehyde dehydrogenase, plastidic pyruvate dehydrogenase complex and ATP-citrate lyase), and compared these with the expression of acetyl-CoA-metabolizing genes (heteromeric and homomeric acetyl-CoA carboxylase). These comparisons have led to the development of testable hypotheses as to how distinct pools of acetyl-CoA are generated and metabolized. These hypotheses are being tested by combined biochemical, genetic and molecular biological experiments, which is providing insights into how acetyl-CoA metabolism is regulated.     

Acetyl-CoA provision and the acetyl group deficit at the onset of contraction in ischemic canine skeletal muscle

We examined the effects of increasing acetylcarnitine and acetyl-CoA availability at rest, independent of pyruvate dehydrogenase complex (PDC) activation, on energy production and tension development during the rest-to-work transition in canine skeletal muscle. We aimed to elucidate whether the lag in PDC-derived acetyl-CoA delivery toward the TCA cycle at the onset of exercise can be overcome by increasing acetyl group availability independently of PDC activation or is intimately dependent on PDC-derived acetyl-CoA. Gracilis muscle pretreated with saline or sodium acetate (360 mg/kg body mass) (both n = 6) was sampled repeatedly during 5 min of ischemic contraction. Acetate increased acetylcarnitine and acetyl-CoA availability (both P < 0.01) above control at rest and throughout contraction (P < 0.05), independently of differences in resting PDC activation between treatments. Acetate reduced oxygen-independent ATP resynthesis approximately 40% (P < 0.05) during the first minute of contraction. No difference in oxygen-independent ATP resynthesis existed between treatments from 1 to 3 min of contraction; however, energy production via this route increased approximately 25% (P < 0.05) above control in the acetate-treated group during the final 2 min of contraction. Tension development was approximately 20% greater after 5-min contraction after acetate treatment than in control (P < 0.05). In conclusion, at the immediate onset of contraction, when PDC was largely inactive, increasing cellular acetyl group availability overcame inertia in mitochondrial ATP regeneration. However, after the first minute, when PDC was near maximally activated in both groups, it appears that PDC-derived acetyl-CoA, rather than increased cellular acetyl group availability per se, dictated mitochondrial ATP resynthesis.     

Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase

The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Km's were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed.     

The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds

Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1alpha- and ptE1beta-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1beta mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1beta mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds.     

Lipogenic enzymes in rat maternal adipose tissue in the perinatal period.

1. The specific activities of fatty acid synthetase, acetyl-CoA carboxylase and pyruvate dehydrogenase were measured in rat adipose-tissue extracts in pregnancy and lactation. Fatty acid synthetase specific activity correlates very closely with the rate of fatty acid synthesis, the enzyme specific activity decreasing after mid-pregnancy in a manner very similar to the rate of fatty acid synthesis. Acetyl-CoA carboxylase specific activity also decreases dramatically after mid-pregnancy. Initial pyruvate dehydrogenase specific activity shows a decrease between 2 days pre partum and 2 days post partum, but total enzyme activity shows no significant change in the same period. 2. Immunotitrations of fatty acid synthetase and pyruvate dehydrogenase activities were carried out; the titrations showed that the change in the fatty acid synthetase activity is due to a change in the enzyme amount; the amount of pyruvate dyhydrogenase does not change. Therefore the decrease in fatty acid biosynthesis in subcutaneous and parametrial adipose tissue in late pregnancy and early lactation is associated with a decrease in the amount of at least one of the enzymes involved in fatty acid biosynthesis. The correlation of these events with known hormonal changes is discussed.     

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pyruvate metabolism by lymphocytes: evidence for an additional ketogenic tissue

The rate of utilization of pyruvate (at various concentrations) was measured in lymphocytes prepared from rat mesenteric lymph nodes. The quantitative contribution of pyruvate to CO2, lactate, aspartate, alanine, citrate, acetate, acetyl-CoA and ketone bodies accounted for the pyruvate metabolized. Pyruvate utilization was depressed by increasing concentrations of pyruvate. The maximum catalytic activities and selected intracellular distributions of the following enzymes of pyruvate, citrate and acetyl-CoA metabolism were measured: citrate synthase, ATP-citrate lyase, lactate dehydrogenase, acetyl-CoA hydrolase, acetylcarnitine transferase, NAD+- and NADP+- isocitrate dehydrogenases, HMG-CoA lyase, HMG-CoA synthase, Pyruvate dehydrogenase, acetoacetyl-CoA thiolase, 3-oxoacid-CoA transferase, 3-hydroxybutyrate dehydrogenase and pyruvate carboxylase. Acetyl-CoA formed from pyruvate did not contribute to the respiratory energy metabolism of resting lymphocytes. Instead acetyl-CoA was converted to acetoacetate by reactions which may favour the pathway catalyzed by acetoacetyl-CoA thiolase and 3-oxoacid-CoA transferase. Acetate, acetyl- and palmitoyl-carnitine inhibited the decarboxylation of [1-14C] pyruvate. These observations may be connected with the suppression of pyruvate utilization by increased pyruvate substrate concentration. Only very small amounts of either pyruvate or acetate were incorporated into lipids in resting lymphocytes. The amounts incorporated were partitioned in approximately the same pattern into FFA, T.G., cholesterol and cholesterol esters. Taken together the data show that pyruvate metabolism is directed inter alia at the formation of acetoacetate which may serve as a lipid synthesis precursor. When pyruvate utilization and metabolism was enhanced by concanavalin A, then acetoacetate formation was not favoured and from this it is proposed that the acetyl units may then be directed into lipid synthesis and may also make a contribution to the energy metabolism of the activated lymphocyte.     

Effect of glutamate on the control of fatty-acid synthesis in white adipose tissue of the rat. Inhibition of pyruvate dehydrogenase.

Glutamate (5mM) inhibited glucose conversion to fatty acids by approximately one-third in adipocytes from fed rats. This inhibition was significantly less in the pressence of pyruvate or 2-oxoglutarate. After incubation of adipose tissue from fed rats with glucose and insulin, pyruvate dehydrogenase activity was 180 plus or minus 17 mU/g wet weight. Addition of glutamine to the incubation medium decreased this activity significantly (118 plus or minus 14 mU/g wet weight). This inhibition by glutamate was also diminished when 2-oxoglutarate or pyruvate were present. Glutamate added to homohentates of adipose tissue had no effect on the activation of pyruvate dehydrogenase by Mg-2+. However, glutamate inhibited the active form of the enzyme and enhanced the rate of inactivation of the enzyme complex by ATP and Mg-2+. Aminooxyacetate, a transaminase inhibitor, did not reverse the effects of glutamate on pyruvate dehydrogenase nor fatty acid synthesis.     

Acetyl-CoA synthetase (ADP forming) in archaea,a novel enzyme involved in acetate formation and ATP synthesis

The anaerobic hyperthermophilic archaea Desulfurococcus amylolyticus, Hyperthermus butylicus, Thermococcus celer, Pyrococcus woesei, the hyperthermophilic bacteria Thermotoga maritima and Clostridium thermohydrosulfuricum and the aerobic mesophilic archaeon Halobacterium saccharovorum were grown either on complex media, on sugars or on pyruvate as carbon and energy sources. During growth acetate was formed as fermentation product by all organisms. The enzymes involved in acetyl-CoA formation from pyruvate and in acetate formation from acetyl-CoA were investigated:

1. Cell extracts of all species, both archaea and bacteria, catalyzed the coenzyme A-dependent oxidative decarboxylation of pyruvate with viologen dyes or with Clostridium pasteurianum ferredoxin as electron acceptors indicating a pyruvate: ferredoxin oxidoreductase to be operative in acetyl-CoA formation from pyruvate.
2. Cell extracts of all archaeal species, both hyperthermophiles (D. amylolyticus, H. butylicus, T. celer, P. woesei) and the mesophile H. saccharovorum, contained an acetyl-CoA synthetase (ADP forming), which catalyzes both acetate formation from acetyl-CoA and ATP synthesis from ADP and phosphate (P_i): Acetyl-CoA+ADP+P_i?Acetate + ATP+CoA. Phosphate acetyltransferase and acetate kinase could not be detected.
3. Cell extracts of the hyperthermophilic (eu)bacteria T. maritima and C. thermohydrosulfuricum contained phosphate acetyltransferase and acetate kinase rather than acetyl-CoA synthetase (ADP forming).

These data indicate that acetyl-CoA synthetase (ADP forming) represents a typical archaeal property rather than an enzyme specific for hyperthermophiles. It is proposed that in all acetate forming archaea the formation of acetate and of ATP from acetyl-CoA, ADP and P_i are catalyzed by acetyl-CoA synthetase (ADP forming), whereas in all acetate forming (eu)bacteria these reactions are catalyzed by two enzymes, phosphate acetyltransferase and acetate kinase.     

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae.

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acetate-Activating Enzymes of Bradyrhizobium japonicum Bacteroids

Acetyl coenzyme A (acetyl-CoA) synthetase and acetate kinase were localized within the soluble portion of Bradyrhizobium japonicum bacteroids, and no appreciable activity was found elsewhere in the nodule. The presence of each acetate-activating enzyme was confirmed by separation of the two enzyme activities on a hydroxylapatite column, by substrate dependence of each enzyme in both the forward and reverse directions, by substrate specificity, by inhibition patterns, and also by identification of the reaction products by C₁₈ reverse-phase high-pressure liquid chromatography. Phosphotransacetylase activity, found in the soluble portion of the bacteroid, was dependent on the presence of potassium and was inhibited by added sodium. The greatest acetyl-CoA hydrolase activity was found in the root nodule cytosol, although appreciable activity also was found within the bacteroids. The combined specific activities of acetyl-CoA synthetase and acetate kinase-phosphotransacetylase were approximate to that of the pyruvate dehydrogenase complex, thus providing B. japonicum with sufficient capacity to generate acetyl-CoA.     

The effect of progesterone on prolactin stimulation of fatty acid synthesis, glycerolipid synthesis and lipogenic-enzyme activities in mammary glands of pseudopregnant rabbits, after explant culture or intraductal injection.

The activities of lipogenic enzymes, such as acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase, and glycerolipid synthesis increased significantly in mammary explants of 11-day-pseudopregnant rabbits in response to prolactin, in the presence of near-physiological concentrations of insulin and corticosterone in culture. Increasing the concentration of progesterone in culture resulted in suppression of glycerolipid synthesis and activities of acetyl-CoA carboxylase and fatty acid synthetase, but not the pentose phosphate dehydrogenases. However, at near-physiological concentration of progesterone, only acetyl-CoA carboxylase activity was decreased. Injection of prolactin intraductally into 11-day-pseudopregnant rabbits stimulated glycerolipid synthesis, fatty acid synthesis and enzymes involved in fatty acid synthesis, after 3 days. Intraductal injection of progesterone separately or together with prolactin had no significant effect on basal or stimulated lipogenesis in mammary glands. Intramuscular injection of progesterone at 10 mg/day did not suppress fatty acid synthesis stimulated when prolactin was injected intraductally, but a significant inhibition was observed at a higher dose (80 mg/day).     

Fatty-acid synthesis in chloroplasts from mustard (Sinapis alba L.) cotyledons: formation of acetyl coenzyme A by intraplastid glycolytic enzymes and a pyruvate dehydrogenase complex

Chloroplasts from the cotyledons of mustard (Sinapis alba L.) seedlings were isolated on Percoll gradients, and showed a high degree of intactness (92%) and purity as judged by electron microscopy and marker-enzyme analysis (cytoplasmic contamination lower than 0.4% on a protein basis). The chloroplasts synthesized longchain fatty acids from both precursors [1-¹⁴C] acetate and [2-¹⁴C]pyruvate; maximum incorporation rates were 96 nmol·(mg Chl)^-1·h^-1 for acetate and 213 nmol·(mg Chl)^-1·h^-1 for pyruvate. Acetyl-CoA-producing enzymatic activities, namely acetyl-CoA synthetase (EC 6.2.1.1.) and a pyruvate dehydrogenase complex, showed specific activities of 14.8 nmol·(mg protein)^-1·min^-1 and 18.2 nmol·(mg protein)^-1·min^-1, respectively. The glycolytic enzymes phosphoglyceromutase (EC 2.7.5.3) phosphopyruvate hydratase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were all found to be components of these chloroplasts, thus indicating a possible pathway for intraplastid acetyl-CoA formation.Abbreviations ACS acetyl coenzyme A synthetase - Chl chlorophyll - DTE 1,4-dithioerythritol - PDHC pyruvate dehydrogenase complex - 3-PGA 3-phosphoglyceric acid