Regulation of the biosynthesis of cytochrome P-450 in brewer's yeast. Role of cyclic AMP.

The drug metabolising enzyme cytochrome P-450 has been studied in great detail in mammalian systems and its presence in microorganisms is also well established. However, neither its function nor its means of control in brewer's yeast, Saccharomyces cerevisiae, has been investigated. We demonstrate here using yeast protoplasts that it is the intracellular concentration of cyclic AMP which controls, by repression, the de novo synthesis of the enzyme, and also that cyclic AMP concentrations are in turn inversely related to the concentration of glucose in the yeast growth medium.     

Cyclic AMP,fructose-2,6-bisphosphate and catabolite inactivation of enzymes in the hydrocarbon-assimilating yeast Candida maltosa

The inactivation of fructose-1,6-bisphosphatase, isocitrate lyase and cytoplasmic malate dehydrogenase in Candida maltosa was found to occur after the addition of glucose to starved cells. The concentration of cyclic AMP and fructose-2,6-bisphosphate increased drastically within 30 s when glucose was added to the intact cells of this yeast. From these results it was concluded that catabolite inactivation, with participation of cyclic AMP and fructose-2,6-bisphosphate, is an important control mechanism of the gluconeogenetic sequence in the n-alkane-assimilating yeast Candida maltosa, as described for Saccharomyces cerevisiae.     

Cyclic AMP and the control of aggregative phase gene expression in Dictyostelium discoideum.

The induction of aggregative phase functions and the acceleration of the onset of aggregation competence by nanomolar pulses of cyclic AMP can be mimicked by exposing developing cells to a high extracellular concentration of either cyclic AMP or cyclic GMP (5 × 10^?4M) during the first 1–2 hr of development. Pulses of cyclic AMP have previously been shown to result in oscillations of intracellular cyclic AMP concentration; we show that high extracellular concentrations of cyclic AMP and cyclic GMP cause intracellular cyclic AMP levels to increase. We describe a mutant, HM11, which has elevated levels of intracellular cyclic AMP from the beginning of development and which begins to accumulate cell-associated phosphodiesterase, an aggregative phase enzyme, within an hour of starvation. Our data suggest that the expression of aggregative phase functions is controlled by an elevation of intracellular cyclic AMP which may be either continuous or periodic.     

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.     

Purification and properties of adenosine 3′,5′-monophosphate phosphodiesterase from baker's yeast

Cyclic AMP phosphodiesterase from Saccharomyces cerevisiae was purified about 20,000-fold to homogeneity. The purified enzyme had a molecular weight of about 60,000 as estimated by gel filtration.The enzyme activity was optimal at pH 8.5–9.0 and was not stimulated by imidazole. Among cyclic 3′,5′-nucleotides, cyclic AMP was the most active substrate for the purified enzyme (K_m = 0.25 mM), but it was inhibitory at concentrations above 4 mm. N⁶,O^2′-dibutyryl cyclic AMP was not hydrolyzed at all.Unlike other cyclic AMP phosphodiesterases from various sources, the purified yeast enzyme did not require divalent metal ions for maximal activity and was rather inhibited in various degrees by added metal ions. The enzyme was not very sensitive to thiol inhibitors.The purified yeast enzyme was strongly inhibited by theophylline and slightly by caffeine. In contrast to the enzyme from S. carlsbergensis, the enzyme from S. cerevisiae was not inhibited at all by ATP or PP_i.The enzyme activity was not released into the growth medium, and the intracellular distribution studies indicated that the enzyme was located mainly in the cytosol fraction.     

Species-Specific Aggregation Factor In Sponges

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA -initiation phase the cyclic AMP - and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10⁶ cells to 0.3 pmol/10⁶ cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10⁶ cells. the activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. the soluble as well as the particulate enzyme activities were checked in vitro. the cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

The influence of light and different ATP concentrations on cell aggregation in cyclic AMP sensitive and insensitive species of the cellular slime molds

The influence of light and different concentrations of ATP on cell aggregation in cyclic AMP sensitive (Dictyostelium mucoroides, D. purpureum) and cyclic AMP insensitive species (Polysphondylium violaceum, P. pallidum, D. lacteum) of the cellular slime molds was observed in small and in large amoebal populations.Both light and ATP (optimal concentration:10^-5M) accelerated cell aggregation and increased the number of aggregating centers in large populations. For cyclic AMP sensitive species the effect of ATP in large populations was more pronounced than for the species that do not react to cyclic AMP.A possible explanation for the similar effect of light and ATP has been discussed.     

Activation of RAT erythrocyte phosphofructokinase by AMP and by non-physiological concentrations of Cyclic AMP

Summary Cyclic AMP (300µ m) activates phosphofructokinase from dialyzed haemolysates of mature rat erythrocytes. The main conclusions are: a) Cyclic AMP, at pH 7.1 and low concentrations of fructose-6-phosphate, is able to reverse the inhibition produced by different amounts of ATP (up to 1.5mm). b) The cyclic nucleotide is a positive allosteric effector of the enzyme as shown by the displacement of sigmoidal fructose-6-phosphate saturation curve to hyperbolic kinetics in the presence of inhibitory concentrations (1.5mm) of ATP. c) Cyclic AMP has no significant influence as deinhibitor of phosphofructokinase either at pH 7.1 and non-inhibitory levels (0.25mm) of ATP or at pH 8.1 and inhibitory (1.5mm) of non-inhibitory (0.25mm) concentrations of ATP. Similar conclusions were obtained with 300µ m AMP but not at a lower concentration (3µ m) with both nucleotides.The comparison of cyclic AMP results with those obtained under similar concentrations of AMP suggest that cyclic AMP is really only an in vitro modulator of the enzyme from rat erythrocytes, presumably at an AMP regulatory site, since non-physiological concentrations are required to act as deinhibitor.     

Adaptive mechanisms of the noradrenergic cyclic AMP generating system in the limbic forebrain of the rat: adaptation to persistent changes in the availability of norepinephrine (NE).

Abstract— The noradrenergic cyclic AMP generating system in slices of the limbic forebrain of rats displays characteristics which are compatible with those of a central NE receptor. The cyclic AMP response to a K_max concentration of NE (concentration of NE which elicits maximal increase in the level of cyclic AMP) is significantly enhanced in slices from reserpinized animals, although the K_a value of NE (concentration of NE eliciting half-maximum response) was not significantly changed. Chemosympathectomy with 6-hydroxydopamine (6-OHDA) significantly enhanced the activity of the system to NE and isoproterenol but not to adenosine and reduced the K_a value for NE. The changes in the reactivity of the cyclic AMP generating system following 6-OHDA administration appear to be related to a decrease in the availability of NE and not to that of other neurotransmitters as protection by desipramine (DMI) of noradrenergic neurons against the neurotoxic action of 6-OHDA prevented the development of supersensitivity to NE. Conversely, and independent of the actual concentration of NE in brain tissue, a persistent increase in the availability of NE caused by prolonged MAO inhibition lead to a marked decrease in the reactivity of the cyclic AMP generating system. The results provide further evidence for a regulatory mechanism in the CNS involving the noradrenergic receptor that adapts its sensitivity to NE in a manner inversely related to the degree of its stimulation by the catecholamine.     

The role of cyclic AMP in parathyroid hormone action in the toad bladder

Summary Parathyroid hormone (PTH) inhibited active transport of inorganic phosphate and stimulated an increase in cyclic AMP concentration in the urinary bladder of the toad,Bufo marinus. Active transport of phosphate in the toad bladder was also inhibited by an analog of cyclic AMP (dibutyryl cyclic AMP) and by other drugs (pitressin and theophylline) which increase toad bladder intracellular cyclic AMP concentration. These data support the concept that cyclic AMP may be the mediator of PTH-induced phosphate transport inhibition in the toad bladder.     

The pH and temperature dependence of the activity of the high Km cyclic nucleotide phosphodiesterase of bakers' yeast

The hydrolysis of adenosine 3':5'-monophosphate by the high Km cyclic nucleotide phosphodiesterase of bakers' yeast was studied over a range of temperature and pH at I = 0.17. The effects of ionic strength and MgCl2 concentration were studied at pH 7.7 and 30 degrees C. Km and Vmax were insensitive to changes in the MgCl2 concentration between 1 and 30 mM, implying that this enzyme (which does not require free divalent metal ions) does not discriminate between free cyclic AMP- and the Mg-cyclic AMP+ complex. Vmax decreased below pH 6.8 because of protonation of a group required in the basic form in the enzyme x substrate complex. On the basis of its pK (5.46 at 30 degrees C) and delta H (23 kJ/mol) this group was tentatively identified as imidazole. Vmax/Km decreased above pH 6.8 because of ionization of a group required in the acid form in the free enzyme, with a pK of 7.88 at 30 degrees C and a delta H of about 13 kJ/mol. Several possibilities exist for the identity of this group, the most likely being a second imidazole, sulfhydryl, or a water molecule bonded to tightly bound zinc. At pH 7.90, log Vmax and log Km both changed linearly with 1/T (between 12 degrees C and 37 degrees C) with enthalpies of 47 and 55 kJ/mol, respectively. Consequently, at low enough cyclic AMP concentration, the rate of reaction at pH 7.90 decreases slightly when the temperature is increased. This is also true at higher pH, but in the physiological pH range (6.4 to 7.5) Vmax/Km and, therefore, the rate of reaction at very low cyclic AMP concentration were nearly independent of temperature. Under physiological conditions, the Km approaches the upper limit of in vivo cyclic AMP concentrations in yeast, and at normal in vivo cyclic AMP concentrations the pH optimum is within or below the physiological range of pH in yeast.     

Cyclic AMP,Transmitter Release and the Effect of Adenosine on Neuromuscular Transmission

THE high activities of the enzymes in nervous tissue regulating the metabolism of 3',5' adenosine monophosphate (cyclic AMP) and its apparent involvement in secretion from glands have prompted considerable speculation about its possible role in the release of transmitters from nerve endings^1–4. There is some evidence that cyclic AMP is concerned in the release of acetylcholine from motor nerve endings^1,3. It has been found, for example, that neuromuscular transmission is facilitated by catecholamines^5,6 and methylxanthines^1,3; noradrenaline is known to increase both the quantal content of the endplate potential⁶ and the level of cyclic AMP in nervous tissue^7,8 and the increase in the quantal content is more marked in the presence of theophylline³. It was also found that increases in cyclic AMP concentration of up to thirty-fold occur in brain slices in the presence of 0.1-1 mM adenosine. We have therefore examined the effect of this substance on transmitter release.     

The mechanism by which glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces cerevisiae. A cyclic-AMP-dependent activation of phosphofructokinase 2

When glucose was added to a suspension of Saccharomyces cerevisiae in stationary phase, it caused a transient increase in the concentration of cyclic AMP and a more persistent increase in the concentration of hexose 6-phosphate and of fructose 2,6-bisphosphate. These effects of glucose on cyclic AMP and fructose 2,6-bisphosphate but not that on hexose 6-phosphate were greatly decreased in the presence of 0.15 mM acridine orange or when a temperature-sensitive mutant deficient in adenylate cyclase was used at the restrictive temperature. Incubation of the cells in the presence of dinitrophenol and in the absence of glucose increased the concentration of both cyclic AMP and fructose 2,6-bisphosphate, but with a minimal change in that of hexose 6-phosphate. Glucose induced also in less than 3 min a severalfold increase in the activity of 6-phosphofructo-2-kinase and this effect was counteracted by the presence of acridine orange. When a cell-free extract of yeast in the stationary phase was incubated with ATP-Mg and cyclic AMP, there was a 10-fold activation of 6-phosphofructo-2-kinase. Finally, the latter enzyme was purified 150-fold and its activity could then be increased about 10-fold upon incubation with ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase. This activation resulted from a 4.3-fold increase in V and a 2-fold decrease in Km. Both forms of the enzyme were inhibited by sn-glycerol 3-phosphate. From these results it is concluded that the effect of glucose in increasing the concentration of fructose 2,6-bisphosphate in S. cerevisiae is mediated by the successive activation of adenylate cyclase and of cyclic-AMP-dependent protein kinase and by the phosphorylation of 6-phosphofructo-2-kinase by the latter enzyme. In deep contrast with what is known of the liver enzyme, yeast 6-phosphofructo-2-kinase is activated by phosphorylation instead of being inactivated.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production

The ability of prostaglandin I₂ (PGI₂) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE₂. However, a concentration of 15 μg/ml PGI₂ was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 μg/ml PGE₂, which produced the maximum response. It therefore appears that PGI₂ is not more effective than PGE₂ in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI₂ appeared to be able to modify the stimulation of cyclic AMP production by follicle- stimulating hormone (FSH), but whether or not PGI₂ plays any role in follicular function remains to be established.     

Studies on adenosine 3′,5′-monophosphate phosphodiesterase of human erythrocyte membranes

In this work we show the existence of cyclic AMP phosphodiesterase (EC 3.1.4.17) in human erythrocyte membranes and have clarified some properties of the enzyme. In human erythrocytes, about 23% of the total cyclic AMP phosphodiesterase activity is in a membrane-bound form. Although it could be solubilized with Triton X-100 in 5 mM Tris-HCl buffer (pH 8.0), it was not solubilized by a low or high concentration of salt. The enzyme seems to be localized in the cytoplasmic surface, since it is detected in sealed inside-out vesicles of human erythrocyte membranes, but not in intact human erythrocytes. The optimum pH was found to lie between 7.4 and 8.0, and Mg²⁺ was found to be necessary for its activity. Ca²⁺ and calmodulin could not stimulate the activity of this enzyme. Theophylline was a strong inhibitor, but cyclic GMP could not inhibit the enzymic hydrolysis of cyclic [³²P]AMP and this membrane-bound enzyme therefore seems to be specific to cyclic AMP.     

Biochemical and physiological investigations on adenosine 5ʹ monophosphate deaminase from Plasmodium spp.

The interplay between ATP generating and utilizing pathways in a cell is responsible for maintaining cellular ATP/energy homeostasis that is reflected by Adenylate Energy Charge (AEC) ratio. Adenylate kinase (AK), that catalyzes inter‐conversion of ADP, ATP and AMP, plays a major role in maintaining AEC and is regulated by cellular AMP levels. Hence, the enzymes AMP deaminase (AMPD) and nucleotidases, which catabolize AMP, indirectly regulate AK activity and in‐turn affect AEC. Here, we present the first report on AMPD from Plasmodium, the causative agent of malaria. The recombinant enzyme expressed in Saccharomyces cerevisiae was studied using functional complementation assay and residues vital for enzyme activity have been identified. Similarities and differences between Plasmodium falciparum AMPD (PfAMPD) and its homologs from yeast, Arabidopsis and humans are also discussed. The AMPD gene was deleted in the murine malaria parasite P. berghei and was found to be dispensable during all stages of the parasite life cycle. However, when episomal expression was attempted, viable parasites were not obtained, suggesting that perturbing AMP homeostasis by over‐expressing AMPD might be lethal. As AMPD is known to be allosterically modulated by ATP, GTP and phosphate, allosteric activators of PfAMPD could be developed as anti‐parasitic agents.     

Studies on Metabolic Pathway of NAD in Yeast

The enzyme system of NAD degradation was extracted from autolysate of Saccharomyces oviformis.

The enzymes were partially separated by ammonium sulfate fractionation and DEAE-cellulose column chromatography, and then the metabolic pathway of NAD in yeast was presented, in which four enzymes were contained. It has been found that, among them, the 5?nucleotidase has more affinity for AMP and the nucleosidase has strict affinity for nicotinamide riboside.

In the degradation of NAD in the yeast, nucleotide pyrophosphatase was main enzyme, but NADase, nucleotide pyrophosphorylase and adenosine deaminase seemed not to play an important role.     

Catabolite inactivation,cyclic AMP and protein phosphorylation in the methylotrophic yeastHansenula polymorpha

The inactivation of the peroxisomal enzyme alcohol oxidase and the cytoplasmic enzymes fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase was found to occur after addition of glucose to methanol-grown cells of the yeastHansenula polymorpha. The concentration of cyclic AMP increased nearly twofold within 3 min under the same conditions. In crude extracts ofH. polymorpha about 20 proteins are phosphorylated by cyclic AMP dependent protein kinases, among them also fructose-1,6-bisphosphatase. No phosphorylation of the alcohol oxidase protein could be detected. From this fact, it was concluded that the inactivation of the peroxisomal alcohol oxidase is independent of cyclic AMP-dependent protein phosphorylation.