Gluconeogenetic Enzymes Research Articles

Cloning and analysis of the nuclear gene MRP-S9 encoding mitochondrial ribosomal protein S9 of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae nuclear gene MRP-S9 was identified as part of the European effort in sequencing chromosome II. MRP-S9 encodes for a hydrophilic and basic protein of 278 amino acids with a molecular mass of 32 kDa. The C-terminal part (aa 153-278) of the MRP-S9 protein exhibits significant sequence similarity to members of the eubacterial and chloroplast S9 ribosomal-protein family. Cells disrupted in the chromosomal copy of MRP-S9 were unable to respire and displayed a characteristic phenotype of mutants with defects in mitochondrial protein synthesis as indicated by a loss of cytochrome c oxidase activity. Additionally, no activities of the gluconeogenetic enzymes, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, could be observed under conditions of glucose de-repression. The respiration-deficient phenotype could not be restored by transformation of the disruption strain with a wild-type copy of MRP-S9, indicating that MRP-S9 disruption led to rho- or rho0 cells. Sequence similarities of MRP-S9 to other members of the ribosomal S9-protein family and the phenotype of disrupted cells are consistent with an essential role of MRP-S9 is assembly and/or function of the 30s subunit of yeast mitochondrial ribosomes.

Molecular genetic analysis of glucocorticoid signalling in development

A null mutation of the glucocorticoid receptor was generated by homologous recombination. Mutant newborn mice showed impaired lung development, hypertrophy of the adrenal cortex and a strongly reduced size of the adrenal medulla. Phenylethanolamine N-methyltransferase (PNMT) was undetectable in the adrenals of the mutant mice. Serum levels of corticosterone were moderately and ACTH levels were strongly elevated in the mutants. A weaker but significant increase of corticosterone and ACTH was observed already in heterozygous animals. This points to a dysregulation of the HPA axis due to defective feedback regulation via the glucocorticoid receptor. Liver gluconeogenetic enzymes were reduced to a variable degree. Whereas survival of heterozygous mutants was not affected, most of the homozygous mutant mice died during the perinatal period.

Characterization of a mutant of the yeast Candida maltosa defective in catabolite inactivation of gluconeogenetic enzymes

A spontaneous mutant of the yeast Candida maltosa SBUG 700 was isolated showing pseudohyphal marphology under all growth conditions tested. The C. maltosa PHM mutant takes up glucose with the kinetics of C. maltosa SBUG 700 and starved cells contain the same cyclic AMP concentration. Addition of glucose to the PHM mutant does not result in an increase of the intracellular cyclic AMP level and in catabolite inactivation of fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. However, addition of 2,4-dinitrophenol is followed by a rapid, transient increase of the cyclic AMP level in the mutant cells, but not by catabolite inactivation. These results show that a common mechanism might be responsible for catabolite inactivation and glucose-induced cAMP signaling or that glucose-induced cAMP signaling is required for catabolite inactivation in C. maltosa.

Study of carbohydrate metabolism in glycogen storing cell lines derived from cultured rat hepatocytes

1. 1. Some aspects of carbohydrate metabolism were investigated in three non-malignant, glycogen storing, cell lines derived from a primary culture of rat hepatocytes, and in the Morris hepatoma 3924 cells. 2. 2. The three cell lines show biochemical alterations which are, to a large extent, similar to those found in the hepatoma cells: increased activity of glycolytic enzymes and decreased activity of gluconeogenetic enzymes. 3. 3. An increase of glucose-6-phosphate dehydrogenase activity is also found. 4. 4. The three cell lines, as the Morris hepatoma cells, actively convert glucose into lactate under the in vitro conditions of culture. Fructose is not taken up as quickly as glucose and galactose is not metabolized. 5. 5. As compared with normal hepatocytes, the three cell lines have altered metabolism and growth behaviour. They largely resemble the preneoplastic cells appearing in rat liver at the early stages of experimental carcinogenesis.

Studies on glucose-induced inactivation of gluconeogenetic enzymes in adenylate cyclase and cAMP-dependent protein kinase yeast mutants.

Glucose-induced inactivation of the gluconeogenetic enzymes fructose-1,6-biphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase was tested in yeast mutants defective in adenylate cyclase (cyr1 mutation) and in the cAMP-binding subunit of cAMP-dependent protein kinase (bcy 1 mutation). In the mutant AM7-11D (cyr1 mutation), glucose-induced cAMP overshoot was absent, and no significant inactivation of the gluconeogenetic enzymes was detected, thus supporting the role of cAMP in the process. Moreover, in the mutant AM9-8B (bcy1 mutation), no cAMP-dependent protein kinase activity was evidenced, and, in addition, a normal inactivation pattern was observed, thus indicating that other mechanisms evoked by glucose might be required in the process. In the double mutant AM7-11DR-4 (cyr1 bcy1 mutations), no inactivating effect was triggered by the sugar: this suggests that cAMP exerts some additional effect on the process, besides the activation of cAMP-dependent protein kinase. Furthermore, in AM7-11D, extracellular cAMP triggered about 50% of inactivation of fructose-1,6-bisphosphatase; this effect was largely reversed in acetate medium plus cycloheximide even after 150 min of incubation. However, an extensive and essentially irreversible inactivation was evidenced in the presence of glucose plus cAMP, whereas glucose alone was only slightly effective. Therefore, the reversible effect of cAMP, which probably corresponds to enzyme phosphorylation, seems to be required for the irreversible, probably proteolytic, glucose-stimulated inactivation of this enzyme. Cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase in AM7-11D were also inactivated by cAMP, and much more by glucose plus cAMP, whereas glucose was practically ineffective. However, reversibility of the effect was not detected, and, in addition, no phosphorylation of phosphoenolpyruvate carboxykinase could be evidenced. Therefore, the sugar quite probably stimulates proteolysis of these enzymes, but the mechanism of cAMP in their degradation has still to be defined.

Diminution of ethanol-induced hyperglycaemia in the rat by administration of beta-lactam antibiotics with a tetrazole thiol group

The acute-onset hyperglycaemia produced by i.p. injection of 2 g of ethanol/kg body weight in adult female SPF Sprague-Dawley rats (200-220 g) was considerably less pronounced when 100 mumol/kg b.w. of one of the following tetrazole thiol-containing beta-lactam antibiotics (BLAs) was given i.p. 3 hours in advance: cephamandole (CMD), moxalactam (MOX), cephoperazone (CPZ) or cephothiam (CTM). An equimolar dose of cephazolin (CEZ), a thiadiazole thiol-containing cephalosporin, did not affect the ethanol-induced hyperglycaemia. Administration of an equimolar oral dose of disulfiram, which has an NCS structure similar to that of the tetrazole thiol-containing BLAs, produced an additional increase of the ethanol-induced hyperglycaemia. The diminution of the ethanol-induced hyperglycaemia by BLAs with a tetrazole thiol group appears to be linked to their NCS structure. It is conceivable that this effect which might be important for human therapy, is causally related to an inhibition of the glycolytic or gluconeogenetic enzyme system.

Dependence on cyclic AMP of glucose-induced inactivation of yeast gluconeogenetic enzymes

1. INTRODUCTION In some yeasts, addition of glucose or metabol- ically related sugars to cells adapted to a sugar- lacking medium causes a time-dependent disap- pearance of some enzymes. The proposed physio- logical role of this ‘catabolite inactivation’ is the regulation of glucose formation, since nearly all of the involved enzymes take part in metabolic reac- tions that provide the cells with glucose

Effect of caffeine on glucose-induced inactivation of gluconeogenetic enzymes in Saccharomyces cerevisiae. A possible role of cyclic AMP.

The mechanism of catabolite inactivation of three gluconeogenetic enzymes, fructose-1,6-bisphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase, has been studied in the yeast Saccharomyces cerevisiae. The glucose-induced inactivation of the three enzymes is remarkably retarded by preincubation of the cells with different caffeine concentrations; however, a full conservation of activity has never been obtained, even at the highest drug concentration. Caffeine modifies the metabolic effects produced in the yeast cell by exposure to glucose. It reduces the consumption rate of glucose; changes the glycolytic intermediate pattern, giving rise to a crossover point at the level of the phosphofructokinase/fructose-bisphosphatase cycle; and increases the ATP level and the energy charge. Moreover, it substantially reduces the peak of intracellular cAMP content that immediately follows glucose entry; the magnitude of this effect is dependent on the drug concentration. The effect on the change of intracellular cAMP level appears, among all metabolic effects determined by caffeine, the only plausible one to explain the interference with catabolite inactivation of enzymes. Actually a strong negative correlation between residual activity of each of the three investigated enzymes and intracellular cAMP level has been demonstrated. The existence of a common mechanism of action of cAMP, as the mediating factor for catabolite inactivation of all three enzymes, is proposed.

System identification of the control of gluconeogenesis in the intact organism under conditions of long-term stress.

In this article a system-theoretic approach to hormonal control of gluconeogenesis as observed in the intact organism under hypoglycemia and hypoxia is attempted. Under such conditions the secretion of the stress hormone corticosterone by the adrenal cortex is effected by neural control centres inducing the synthesis of gluconeogenetic enzymes which catalyze the production of glucose from amino acids originating from body tissues. The time courses of the hormonal, enzymatic and metabolic quantities measured are evaluated in the framework of a model in the form of a system of differential equations, the parameters of which are determined by a numerical search procedure.

Formation of Enzymes of Autotrophic Metabolism During Heterotrophic Growth of Alcaligenes eutrophus

SUMMARY: Alcaligenes eutrophus strain H16 formed key enzymes of autotrophic metabolism during heterotrophic growth. The formation of the soluble and membrane-bound hydrogenases, ribulose-5-phosphate kinase and ribulosebisphosphate carboxylase were investigated. In addition, selected enzymes shared by autotrophic and heterotrophic carbon metabolism were examined. Key enzymes of autotrophic metabolism were not detected during exponential growth on succinate, pyruvate or acetate, but were found at intermediate activities in cells grown on fructose, gluconate or citrate. Growth with succinate at a suboptimal pH of 7.7 resulted in a decreased growth rate and a marked increase of enzyme activities. Oxygen-limited growth with succinate also led to a derepression of the synthesis of the hydrogenases and the key enzymes of the Calvin cycle. During growth on glycerol or formate, the activities of these enzymes were comparable with those found under autotrophic conditions with H2 and CO2. The results indicate that both the hydrogenases and the key enzymes of the Calvin cycle were formed under conditions of limited availability of energy. Molecular hydrogen was not required for the formation of the hydrogenases. The regulation of the gluconeogenetic enzymes, common to both autotrophic and heterotrophic carbon metabolism, was more balanced. An increase of enzyme activities was observed under autotrophic conditions, in accord with the physiological role of these enzymes under autotrophic and heterotrophic growth conditions.

Enzyme histochemical studies on the conducting system of the human heart.

In this communication, the results of applying various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases in the human heart are presented. The Purkinje fibres of the atrioventricular conducting system of the human heart differ from the myocardium proper in containing a slightly higher activity of most of the glycolytic and gluconeogenetic enzymes investigated. The relatively higher activity of 6-phosphofructokinase, the key enzyme in anaerobic carbohydrate metabolism, is especially noteworthy. On the other hand, the activities of some of the enzymes that play a part in the aerobic energy metabolism is slightly less than those in the myocardium fibres. As for the activity of the NADPH regenerating enzymes, the activity of 6-phosphogluconate dehydrogenase and malate dehydrogenase (oxaloacetate-decarboxylating) is somewhat higher, and the activity of glucose-6-phosphate dehydrogenase similar, in the Purkinje fibres compared to that in the myocardial fibres. The activity of myosin ATPase is similar for both types of fibre. Likewise, the fibres of the conducting system and of the myocardium show a similar activity of acid phosphatase, beta-glucuronidase, non-specific naphthylesterase and peroxidase. The neurogenic function of the conducting system of the human heart was demonstrated by the high activity of acetylcholinesterase in the Purkinje fibres and in the atrioventricular node. All these histochemical findings in Purkinje fibres are similar at widely differing levels of the conducting system.

The effect of hypoxia on carbohydrate metabolism in flounder ( Platichthys flesus L.)—II. High energy phosphate compounds and the role of glycolytic and gluconeogenetic enzymes

1. 1. ATP, ADP, AMP, energy charge potential and total adenylates in heart, kidney and muscle are relatively unaffected by environmental hypoxia. In the liver, hypoxia causes a 90% drop in ATP, a rise in ADP and AMP, and a drop in energy charge potential and total adenylates. In the muscle tissue ATP concentration is stabilized by a large creatine phosphate pool. 2. 2. Hexokinase activity in the heart is 20 times higher than in the swimming muscle, and thus the heart has a high potential for utilizing exogenous glucose as an anaerobic substrate. 3. 3. The role of creatine phosphate in regulating muscle glycolysis is discussed on background of the strong inhibition of muscle phosphofructokinase by physiological concentrations of creatine phosphate. 4. 4. Flounder heart has a dominating M-type lactate dehydrogenase which is identical to the muscle enzyme by electrophoretic and kinetic criteria. This improves the anaerobic capabilities of the flounder heart compared to other fish hearts. 5. 5. Both liver and kidney have high activities of the gluconeogenetic enzymes glucose-6-phosphatase, fructose-1,6-diphosphatase, and phosphoenolpyruvate carboxykinase and both are capable of synthesizing glucose from [ 14C]lactate. Because of more favorable energy conditions in the kidney this organ may substitute the liver as a gluconeogenetic organ during hypoxia.

Changes in some carbohydrate metabolizing enzymes and glycogen in liver, glucose and lipid in serum during vitellogenesis and after induction by estradiol-17-β in the flounder ( Platichtys flesus L.)

1. 1. Parallel with the increase in the concentration of vitellogenin in serum in nature, (October-November) the activity of the gluconeogenetic enzymes FDP-ase and G-6-P-ase decreases and the glycolytic enzymes PK and PFK increases. 2. 2. In the spawning period in January to May the activity of PFK and G-6-PDH decreases, and the activity of FDP-ase increases. 3. 3. Estradiol-treatment of the males levels off the sex-bound differences in the measured carbohydrate metabolizing enzymes compared to the natural vitellogenic female and increases the activity of the carbohydrate metabolizing enzymes in both sexes. 4. 4. Estradiol-treatment increases the serum concentrations of vitellogen and phospholipid in both sexes and decreases the glucose concentration in the male flounder.

Selective modification of fructose 1,6-bisphosphatase by periodate-oxidized AMP

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Enzyme histochemical studies on the Purkinje fibers of the atrioventricular system of the bovine and porcine hearts.

In this communication the results of applying various histochemical semipermeable membrane techniques to the localization of several enzymes in bovine and porcine heart are presented. The Purkinje fibers of the atrioventricular conducting system of the bovine heart differ from the myocardium proper in containing a greater activity of the glycolytic and gluconeogenetic enzymes--lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase, hexokinase, glucosephosphate isomerase and phosphoglucomutase, and less activity of the aerobic enzymes--NADH: nitroBT oxidoreductase and isocitrate dehydrogenase (NADP+). The metabolic reactions obtained with Purkinje fibers of the porcine heart are less pronounced. These histochemical findings are in accordance with the impression that Purkinje fibers, compared with the common myocardial fibers, have a higher rate of anaerobic metabolism and a lower rate of aerobic metabolism. The activity of the NADPH regenerating enzymes glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating), and the activity of acid hydrolases such as non-specific esterase and acid phosphatase is higher in the Purkinje fibers of both the bovine and porcine heart.

A yeast mutant with glucose-resistant formation of mitochondrial enzymes

Yeast mutants with glucose-insensitive formation of mitochondrial enzymes were isolated starting with a strain completely lacking alcohol dehydrogenase activity. The mutations could uniquely be attributed to a single nuclear gene, designated CCR80. They were largely dominant. Glucose-resistant enzyme formation was most prominent with regard to mitochondrial enzymes succinate dehydrogenase and NADH: cytochrome c oxidoreductase. The effect of CCR80r mutations was rather small but significant on the gluconeogenetic enzymes isocitrate lyase, malate synthase and fructose-1,6-bisphosphatase and on invertase synthesis. The repressive effect of maltose in CCR80r mutants was also reduced showing that glucose-resistance is not caused by a mere hexose uptake defect. This regulatory disorders were not accompanied by reduced levels of glycolytic enzymes or drastically altered levels of glycolytic intermediates. Aerobic fermentation of glucose was almost completely inhibited in the mutants; anaerobic glucose degradation was reduced but not completely abolished. Therefore, the mutants appear to be altered in the regulation of glycolysis. A largely glucose-resistant synthesis of respiratory enzymes is obviously a corollary of this alteration.

Alteration of the maximal activity of the gluconeogenetic enzyme fructose-1,6-diphosphatase of skeletal muscle by cross-reinnervation: A histochemical and biochemical investigation of fatiguability-related aspects

Fatiguability is a determining characteristic of different muscle fibre types. An important aspect, indirectly related to fatiguability, gluconeogenesis, was investigated by observing fructose-1,6-diphosphatase (FDP) activity in experimental models prepared in rabbits by cross-reinnervation of the fatigue-resistant m.soleus (postural muscle) and the highly fatiguable m.flexor digitorum longus (fast muscle — m.fdl). The resultant reprogramming of the m. soleus was associated with greatly intensified FDP activity. Changes in the m.fdl 6–9 months after cross-reinnervation indicated a shift in the opposite direction. The study adds some data on the much neglected state of fatiguability to the otherwise much explored field of alien reinnervation.

On the Activity and Regulation of Anaplerotic and Gluconeogenetic Enzymes during the Growth Process of Baker's Yeast

The anaplerotic and gluconeogenetic metabolism of baker's yeast was studied at the enzymatic level during glucose-ethanol diauxic growth in the presence and absence of aspartate. Of the two possible anaplerotic systems, only the pyruvate carboxylase by-pass was present during the whole growth process. The second system, the glyoxylate by-pass (isocitrate lyase as the indicator), like the specific enzymes of the gluconeogenetic metabolism, phosphoenolpyruvate carboxykinase and hexosediphosphatase began to appear only after the glucose had been consumed. The addition of glucose during the growth phase based on ethanol effected a rapid disappearance of phosphoenolpyruvate carboxykinase and hexosediphosphatase activities. The activity of pyruvate carboxylase decreased when the growth medium was supplied with asparate. The presence of aspartate had no effect on the activities of the other enzymes studied.

Effect of aeration on the activity of gluconeogenetic enzymes in Saccharomyces cerevisiae growing under glucose limitation

1. The effect of aeration on the key enzymes of gluconeogenesis was studied in baker's yeast (Saccharomyces cerevisiae) and in a nonrespiratory variant of S. cerevisiae grown under glucose limitation. 2. In baker's yeast phosphoenolpyruvate carboxykinase, hexosediphophatase and isocitrate lyase were completely repressed under anaerobic conditions. Their repression could be partially reversed by using intense aeration. 3. In the nonrespiratory variant these enzymes were absent independently of aeration. 4. Pyruvate carboxylase of baker's yeast showed maximal activity under anaerobic conditions. In the nonrespiratory variant pyruvate carboxylase had low activity under both anaerobic and aerobic conditions.

Ethanol-Induced Inhibition of Human Intestinal Enzyme Activities: Reversal by Folic Acid

The effects of ethyl alcohol on intestinal enzyme activities were studied in 10 normal volunteer subjects consuming either a constant, synthetic diet containing 100 gg of folic acid per day, or an ad libitum mixed diet. During the constant diet, ethanol (15 ml four times a day) caused a 36 to 45% decrease in the activities of the glycolytic enzymes, hexokinase, fructose-l-phosphate aldolase, fructose-1, 6-diphosphate aldolase, and a 58% decrease in the activity of the gluconeogenetic enzyme fructose-l, 6-diphosphatase. Conversely, the ethanol caused a 33% increase in the activity of pyruvate kinase. Folate given with the ethanol reversed the inhibitory action of ethanol by causing a 44% increase in fructose-l-phosphate aldolase, 42% in fructose-1, 6-diphosphate aldolase, and 45% in hexokinase, and no change in fructose-1, 6-diphosphatase activity. A synergistic effect occurred with pyruvate kinase which showed a maximum (2.8-fold) increase in activity when both folate and ethanol were given. Folic acid alone caused a 1.6- to 2-fold increase in all the enzymes. Mean plasma and red blood cell folate levels were 1.7 and 116 ng per ml, respectively, at the beginning of the study, and increased to 34.3 and 196 ng per ml, respectively, after folate treatment. Ethanol treatment did not interfere with the absorption of folate when large doses of 15 mg per day were administered, and at no time during the study did folate levels decrease below control values. With the ad libitum mixed diet, changes in enzyme activities with ethanol and folate were qualitatively similar to those seen during the constant synthetic diet. The effect of ethanol and folic acid on intestinal enzyme activities demonstrates how ethanol and folic acid may interact by affecting mutually susceptible enzymes.