Cellular Localization of Acetyl-Coenzyme A Synthetase in Yeast

In cells of Saccharomyces cerevisiae grown with glucose in standing cultures, the microsomal fraction had the highest specific activity for acetyl-coenzyme A synthetase and contained the greatest fraction of the total activity regardless of when the cells were harvested during growth. The addition of acetate did not affect the distribution of the enzyme, nor did subsequent aeration of such cells in phosphate buffer even in the presence of glucose, acetate, or succinate. In cells grown aerobically, however, the microsomal fraction had the highest specific activity and the greatest fraction of the total activity only until the cells reached the stationary phase. After this time, most of the activity was associated with the mitochondrial fraction. Finally, 3 or 4 days after inoculation, this fraction appeared to lose most of the enzyme to the microsomal and soluble fractions. Chloramphenicol, at concentrations that interfered with respiration but not with fermentation, prevented the association of acetyl-coenzyme A synthetase with the mitochondrial fraction in aerated cells, but it did not appreciably affect the large increases in enzyme activity observed during aerobic incubation. Cells grown with glucose under strict anaerobic conditions contained barely detectable amounts of acetyl-coenzyme A synthetase.     

Effect of glucose and cyclic adenosine 3',5'-monophosphate on the synthesis of succinate dehydrogenase and isocitrate lyase in Escherichia coli.

Repressed respiration of Escherichia coli cells grown in the presence of 2% glucose was derepressed when the cells were incubated in a buffer containing casamino acids. The glucose-repressed cells were deficient in succinate dehydrogenase [EC 1.3.99.1] and isocitrate lyase [EC 4.1.3.1] activities, which increased during the incubation. The increases in respiratory activity and enzyme activity on incubation were repressed by glucose, but except for isocitrate lyase these repressions could be restored by the addition of cyclic adenosine 3',5'-monophosphate. Inhibitors of protein synthesis blocked the increase of enzyme activity on incubation without glucose, or with glucose and the cyclic nucleotide.     

Effect of sugars on D-arabitol production and glucose metabolism in Saccharomyces rouxii.

The effect of sugars on the production of d-arabitol and on the glucose catabolic pathways was investigated in the osmotrophic yeast Saccharomyces rouxii. The activity of d-arabitol dehydrogenase, which served as a measure of total d-arabitol production, increased when cells were grown in the presence of increasing glucose concentrations. Growth in sucrose had no effect on the enzyme activity. A high intracellular concentration of d-arabitol could be demonstrated when the cells were grown in a 60% glucose medium and could be eliminated by anaerobic growth or growth in the presence of 4 mg of chloramphenicol per ml. A mutant was isolated that would not grow in 60% glucose; although the regulation of d-arabitol dehydrogenase was altered in this strain, the production of d-arabitol was not eliminated. The activity of d-arabitol dehydrogenase followed the growth phases of the parent strain when the cells were preadapted to 30% glucose. If the cells were adapting from 1 to 30% glucose, a large increase in enzyme activity was detected before growth occurred. Protein synthesis was found to be involved in this increase in activity. There was an increased participation of the pentose phosphate pathway when the cells were grown in the presence of increasing glucose concentrations. The mutant strain had only an 11% pentose phosphate pathway participation compared with 20% for the parent strain in glucose. The results suggest that the active pentose phosphate pathway is involved in glucose tolerance by providing a plentiful supply of reduced nicotinamide adenine dinucleotide phosphate which is necessary for cell survival.     

Altered arachidonic acid biosynthesis and antioxidant protection mechanisms in Schwann cells grown in elevated glucose

In cultured Schwann cells, elevated glucose induces alterations in arachidonic acid metabolism that cause a decrease in the content of glycerophospholipid arachidonoyl-containing molecular species (ACMS). This could result from decreased de novo arachidonic acid biosynthesis, or increased arachidonic acid release from phospholipids. Incorporation of radioactive 8,11,14-eicosatrienoic acid into ACMS was lower for cells grown in 30 mm versus 5 mm glucose, consistent with a decrease in delta5 desaturase activity. However, neither basal arachidonic acid release from prelabeled cells nor stimulated generation of arachidonic acid in the presence of the reacylation inhibitor, thimerosal, the phosphotyrosine phosphatase inhibitor, bipyridyl peroxovanadium, or both together, were altered by varying the glucose concentrations, indicating that arachidonic acid turnover did not contribute to ACMS depletion. Free cytosolic NAD+ /NADH decreased, whereas NADP+ /NADPH remained unchanged for cells grown in elevated glucose, implying that decreased desaturase activity is a result of metabolic changes other than cofactor availability. Schwann cells in elevated glucose were susceptible to oxidative stress, as shown by increased malondialdehyde, depleted glutathione levels, and reduced cytosolic superoxide dismutase activity. Glutathione-altering compounds had no effect on ACMS levels, in contrast to N -acetylcysteine and alpha-lipoic acid, which partly corrected ACMS depletion in phosphatidylcholine. These findings suggest that in the Schwann cell cultures, a high glucose level elicits oxidative stress and weakens antioxidant protection mechanisms which could decrease arachidonic acid biosynthesis and that this deficit can be partly corrected by treatment with exogenous antioxidants.     

Regulation of Glutamine Synthetase X. Effect of Growth Conditions on the Susceptibility of Escherichia coli Glutamine Synthetase to Feedback Inhibition

The kinetic properties of Escherichia coli glutamine synthetase are markedly influenced by the manner in which the organism is grown. Enzyme obtained from stationary-phase cells grown on glycerol and glutamate is strongely inhibited by each of the eight feedback effectors known to influence this enzyme; however, the enzyme from log-phase cells grown on glucose and growth-limiting concentrations of NH(4)Cl is stimulated by some of these effectors. Of the growth variables examined, nitrogen source and time of harvest were the most important; carbon source and aeration seemed to have no effect. Two purified enzyme preparations have been obtained from cells grown under two different conditions, designated enzymes I and II for convenience. Enzyme I is stimulated by adenosine 5'-monophosphate, histidine, and tryptophan in the transfer assay, whereas enzyme II is strongly inhibited by all effectors tested. Enzyme I has a higher specific activity in the forward assay in the presence of Mg(++) or Co(++), whereas enzyme II is more active in the presence of Mn(++).     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

1. The ;initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ;maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80-90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65-75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ;low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ;high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.     

sn-Glycerol-3-phosphate dehydrogenase and its interaction with nitrate reductase in wild-type and hem mutant strains of Staphylococcus aureus

Staphylococcus aureus has membrane-associated sn-glycerol-3-phosphate dehydrogenase activity that is strongly activated by detergents. The enzyme can be measured spectrophotometrically in intact cells in assay systems containing lauryldimethylamine oxide (Ammonyx LO). The dehydrogenase activity was located exclusively in the membrane fraction of cells grown with glycerol under aerobic conditions or under anaerobic conditions with the addition of nitrate; there was no evidence of multiple forms. Development of sn-glycerol-3-phosphate dehydrogenase activity was studied with suspensions of cells grown previously under semianaerobic conditions with glucose and nitrate. The wild-type strain rapidly formed the enzyme when incubated with glycerol under aerobic conditions or under semianaerobic conditions in the presence of nitrate. Under similar conditions, suspensions of hem mutant H-14 required the addition of hemin. Induction of the enzyme was strongly repressed by glucose with both organisms. A procedure was established to obtain cells of mutant H-14 with sn-glycerol-3-phosphate dehydrogenase and nitrate reductase activities, but which could not link the systems unless supplemented with hemin. The coupled activity could also be reconstructed in vitro by the addition of hemin to the depleted membranes.     

Induction of microsomal stearyl coenzyme A desaturase in the newly hatched chicks.

The development of the stearyl-CoA desaturase system was studied in newly hatched chicks. The desaturation activity was very low in hepatic microsomes from chick embryos, less than 0.05 nmol of oleate formed min^?1 (mg of protein)^?1. After hatching and feeding, the desaturation activity gradually increased to 4–5 nmol of oleate formed min^?1 (mg of protein)^?1 in 6-day-old chicks. This increase could be prevented by administration of cycloheximide or actinomycin D. Measurement of the microsomal electron transfer components throughout the induction period showed no significant changes in the NADH- or NADPH-specific reductases or in the concentrations of cytochromes b₅ and P-450. However, the activity of the terminal component of the desaturase system (the desaturase enzyme) increased in parallel with the desaturation activity. Supplementing the liver microsomes from chick embryos with isolated desaturase enzyme resulted in the formation of an active desaturation system. It is proposed that the induction of the stearyl-CoA desaturase system during development of newly hatched chicks is dependent on the synthesis of the terminal desaturase enzyme.     

Regulation of hydrogenase activity in enterobacteria.

Proteus vulgaris, Escherichia coli, and Citrobacter freundii cells were devoid of hydrogenase activity when grown on complex medium or minimal medium plus glucose in the presence of saturating levels of dissolved oxygen. Anaerobically grown cells had appreciable hydrogenase activity. Cells grown anaerobically in the presence of CO (an inhibitor of hydrogenase) or nitrate (an electron acceptor) lacked hydrogenase activity. To make hydrogenase essential for anaerobic growth, cells were grown on fumarate, a nonfermentable carbon source. P. vulgaris and C. freundii evolved H2 gas under these conditions, and the hydrogenase-specific activity was 8 to 10 times greater than that in cells grown on glucose. Cell growth was inhibited by CO, and the cells grew but lacked hydrogenase activity when grown in the presence of nitrate. E. coli grew on fumarate plus H2, and the specific activity was five times greater than that in cells grown on glucose. Thus, hydrogenase activity is inducible and is expressed maximally when the enzyme is essential for cellular growth. Under conditions of growth where the enzyme would not be catalytically active, cells contain little active hydrogenase. Under anaerobic conditions where the enzyme is not essential for growth, the level of hydrogenase activity is intermediate.     

Isolation of a stearoyl CoA Desaturase form Tetrahymena thermophila

Cell free preparations of Tetrahymena thermophila contain an enzyme that catalyzes the direct desaturation of stearoyl CoA to octadecenoic acid. The enzyme is associated with the microsomal fraction of the ciliate. Substrate of the enzyme consists of either free stearic acid or stearoyl CoA. Both ATP and CoA are required when free stearate is the substrate and are also highly stimulatory when stearoyl CoA is the substrate. With stearoyl CoA as the substrate, either NADH or NADPH are required for desaturase activity. In presence of ATP and CoA, either NAD or NADP can replace NADH and NADPH. Desaturase activity is optimal when the enzyme is incubated at pH of 7.2 and a temperature of 30-35 degrees C. Highest levels of the stearoyl CoA desaturase are found in stationary phase ciliates grown at 35 degrees C.     

Simultaneous assay of dihydroxyacetone synthase and transketolase in a methylotrophic yeast grown in continuous culture. A cautionary note

The methylotrophic yeast Candida boidinii CBS 5777 was grown in continuous culture under carbon limitation on glucose, glucose plus methanol, and methanol as carbon and energy sources. During adaptation from glucose to methanol there was a rapid rise in the specific activities of triokinase, fructose-1,6-bisphosphatase and dihydroxyacetone synthase, which are key enzymes of the xylulose phosphate cycle of formaldehyde fixation. The specific activity of classical transketolase fell during this adaptation. Extracts from carbon-limited C. boidinii contained an enzyme which catalysed oxidation of NADH when some preparations or ribose 5-phosphate were added, which was not a transketolase. This enzyme activity was dependent on an impurity in such ribose 5-phosphate preparations and can be confused with transketolase activity.     

Biosynthesis and regulation of fructose-1,6-bisphosphatase and phosphofructokinase in Saccharomyces cerevisiae grown in the presence of glucose and gluconeogenic carbon sources.

The mode of synthesis and the regulation of fructose-1,6-bisphosphatase (Fbpase), a gluconeogenic enzyme, and phosphofructokinase (PFK), a glycolytic enzyme, were investigated in Saccharomyces cerevisiae after growth in the presence of different concentrations of glucose or various gluconeogenic carbon sources. The activity of FBPase appeared in the cells after the complete disappearance of glucose from the growth medium with a concomitant increase of the pH and no significant change in the levels of accumulated ethanol. The appearance of FBPase activity following glucose depletion was dependent upon the synthesis of protein. The FBPase PFK were present in glucose-, ethanol-, glycerol-, lactate-, or pyruvate-grown cells; however, the time of appearance and the levels of both these enzymes varied. The FBPase activity was always higher in 1% glucose-grown cells than in cells grown in the presence of gluconeogenic carbon sources. Phosphoglucose isomerase activity did not vary significantly. Addition of glucose to an FBPase and PFK synthesizing culture resulted in a complete loss, followed by a reappearance, of PFK activity. In the presence of cycloheximide the disappearance of glucose and the changes in the levels of FBPase and PFK were decreased significantly. It is concluded that S. cerevisiae exhibits a more efficient synthesis of FBPase after the exhaustion of glucose compared to the activity present in cells grown in the presence of exogenous gluconeogenic carbon sources. Two metabolically antagonistic enzymes, FBPase and PFK, are present during the transition phase, but not during the exponential phase, of growth, and the decay or inactivation of these enzymes in vivo may be dependent upon a glucose-induced protease activity.     

Gluconeogenesis in Saccharomyces cerevisiae: determination of fructose-1,6-bisphosphatase activity in cells grown in the presence of glycolytic carbon sources.

The activity of fructose-1,6-bisphosphatase (FBP), a gluconeogenic enzyme, was determined in wild-type Saccharomyces cerevisiae X2180 grown in the presence of the glycolytic carbon sources, glucose, fructose, and galactose. The activities of phosphofructokinase (PFK), a glycolytic enzyme, and phosphoglucose isomerase (PGI), an enzyme functioning both in glycolysis and gluconeogenesis, were determined for purposes of comparison. A measurable amount of FBP activity was present in 20-h-old cells grown with moderate shaking in 1% glucose-nutrient or minimal medium. This activity increased significantly in 40 and 60-h-old cells. Similar levels of FBP activity were also present in 20-, 40-, and 60-h-old cells grown in 1% fructose-nutrient medium. A higher level of FBP activity was present in 20-h-old cells grown in 1% galactose-nutrient medium than in 20-h-old cells grown in 1% glucose- or fructose-nutrient medium. The FBP activity in glucose- or fructose-grown cells was higher than the corresponding activity in cells grown under similar conditions for 40 and 60 h in the presence of ethanol, a gluconeogenic carbon source. The PFK activity was significantly less in galactose- and ethanol-grown cells. The PGI activity was relatively constant in 20-, 40-, and 60-h-old cells grown in the presence of glucose, fructose, and galactose, but this activity was reduced approximately 50% in ethanol-grown cells. It is concluded from these results that, depending upon the concentration of carbon source and the time of incubation, FBP, a strictly gloconeogenic enzyme, is synthesized by S. cerevisiae grown in the presence of glycolytic carbon sources.     

Isolation of a Stearoyl CoA Desaturase from Tetrahymena thermophila

Cell free preparations of Tetrahymena thermophila contain an enzyme that catalyzes the direct desaturation of stearoyl CoA to octadecenoic acid. The enzyme is associated with the microsomal fraction of the ciliate. Substrate for the enzyme consists of either free stearic acid or stearoyl CoA. Both ATP and CoA are required when free stearate is the substrate and are also highly stimulatory when stearoyl CoA is the substrate. With stearoyl CoA as the substrate, either NADH or NADPH are required for desaturase activity. In the presence of ATP and CoA, either NAD or NADP can replace NADH and NADPH. Desaturase activity is optimal when the enzyme is incubated at a pH of 7.2 and a temperature of 30–35°C. Highest levels of the stearoyl CoA desaturase are found in stationary phase ciliates grown at 35°C.     

The development of nitrate reductase in Chlorella and its repression by ammonium

Summary Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.     

A possible cyclic AMP-mediated regulation of microsomal fatty acyl-CoA desaturation system in Tetrahymena microsomes

Profound alterations in the microsomal fatty acyl-CoA desaturase activities and cyclic AMP production of a unicellular eukaryote, Tetrahymena pyriformis NT-1, originally grown in the glucose-deficient medium, were observed, following the administration of glucose or beta-adrenergic agonists such as epinephrine and isoproterenol. There was a great increase of stearoyl-CoA (delta 8) desaturase activity coincident with a 2-fold decrease of oleoyl-CoA (delta 12) desaturase activity over the first 2 h after administration of these compounds. During this period of time, it was found that the production in vivo of labeled oleic acid from [14C]acetic or [3H]palmitic acid increases 2-fold and the formation in vivo of each labeled linoleic and gamma-linolenic acids drastically decreases. Glucose or beta-adrenergic agonists caused an increase of stearoyl-CoA-stimulated reoxidation rate of NADH-reduced cytochrome b5 but depressed oleoyl-CoA-stimulated reoxidation rate of b5, indicating that both desaturase activities are controlled by the respective terminal components of the desaturase system. A significant and reproducible increase of adenylate cyclase activity and a slight decrease of cyclic AMP phosphodiesterase activity were observed to occur within the first 2 h after the addition of these compounds, when cyclic AMP content in Tetrahymena cell rose by 3-4-fold. Propranolol, a beta-adrenergic blocker, abolished the effects of glucose or beta-adrenergic agonists on the activities of fatty acyl-CoA desaturases and the terminal components as well as cyclic AMP production of cells. These results suggest that glucose and beta-adrenergic agonists may modulate the microsomal fatty acyl-CoA desaturase system in Tetrahymena by acting through the increase of intracellular cyclic AMP content.     

Studies on the regulation of X-prolyl dipeptidyl aminopeptidase activity

The specific activity of X-prolyl-dipeptidyl aminopeptidase in Saccharomyces cerevisiae grown on glucose-containing medium remains constant during exponential growth and increases less than twofold when cells reach the stationary phase. In cells harvested from exponential growth on glucose-containing medium the specific activity of the enzyme is found to be 20-30% lower than the specific activity observed in media without glucose, containing acetate or ethanol as the carbon source. X-Prolyl-dipeptidyl aminopeptidase is not inactivated after the addition of glucose to stationary phase cells. Growth of the yeast on poor nitrogen sources or under nitrogen-starvation results in a three- to fourfold increase in the level of the enzyme.     

Control of isocitrate lyase in Nocardia salmonicolor (NCIB9701).

Nocardia salmonicolor, grown on acetate, commercial D,L-lactate or hydrocarbon substrates, has high isocitrate lyase activities compared with those resulting from growth on other carbon sources. This presumably reflects the anaplerotic role of the glyoxylate cycle during growth on the former substrates. Amongst a variety of compounds tested, including glucose, pyruvate and tricarboxylic acid cycle intermediates, only succinate and fumarate prevented an increase in enzyme activity in the presence of acetate. When acetate (equimolar to the initial sugar concentration) was added to cultures growing on glucose, there followed de novo synthesis of isocitrated lyase and isocitrate dehydrogenase, with increases in growth rate and glucose utilization, and both acetate and glucose were metabolized simultaneously. A minute amount of acetate (40 muM) caused isocitrate lyase synthesis (a three-fold increase in activity within 3 min of addition) when added to glucose-limited continuous cultures, but even large amounts added to nitrogen-limited batch cultures were ineffective. Malonate, at a concentration that was not totally growth-inhibitory (1mM) prevented the inhibition of acetate-stimulated isocitrate lyase synthesis by succinate, but fumarate still inhibited in the presence of malonate. Phosphoenolpyruvate is a non-competitive inhibitor of the enzyme (apparent Ki 1-7 mM). The results are consistent with the induction of isocitrate or a closely related metabolite, and catabolite repression by a C-4 acid of the tricarboxylic acid cycle, possibly fumarate.     

Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei.

A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.     

Carbohydrate-dependent induction of fatty acid synthase in primary cultures of rat hepatocytes

The glucose and insulin-dependent long-term regulation of fatty acid synthase was studied in primary cultures of hepatocytes from adult female rats. Under basic culture conditions, i.e. 5.5 mM glucose and 0.5 nM insulin, the enzyme activity was continuously decreased over 6 days. In the presence of 100 nM insulin this decrease was reduced but it was not prevented. Enhancement of glucose to 20 mM was followed by an increase of the enzyme activity; after 5 days of treatment the activity was three times higher than under basic culture conditions. The simultaneous presence of 20 mM glucose and of 100 nM insulin resulted in a much more pronounced increase of the activity; after 5 days of treatment the activity was eight times higher than under basic culture conditions. The enhancement was prevented by inhibition of glycolysis. This may indicate that the increase of fatty acid synthase was mediated by a metabolite of glucose rather than by glucose itself. The coordinate regulation of fatty acid synthase and of other lipogenic enzymes was specific as demonstrated by comparison with the activity of lactate dehydrogenase and with synthesis and degradation of cytosolic proteins. The enhancement of the enzyme protein, demonstrated by rocket immunoelectrophoresis, was due to an increase in the rate of enzyme synthesis by 600% as well as to a prolongation of the apparent half-life of the enzyme by 50% (45 h).