Relationship of adenosine-3', 5'-monophosphate to other adenine nucleotides in human platelets

Labeled cyclic AMP and other adenine nucleotides in human platelets were distinctly separated by means of thin-layer chromatography. In lysates of human platelets, ATP was decomposed following the major route: ATP→ADP→AMP→IMP→inosine→hypoxanthine. In contrast, cyclic AMP synthesis occurred rapidly following the breakdown of ATP and leveled off after 30–60 min of incubation. Cyclic AMP synthesis in platelet lysates was 1.02 ± 0.39 nanomoles/hr/mg protein. The level of cyclic AMP formed was related to the 5′-AMP level, although the former did not exceed 5 % of the latter.     

A new chromatographic method using immobilized acriflavin for measuring cyclic AMP in cells prelabeled with radioactive adenine.

A method using the principle of charge-transfer chromatography has been developed for the determination of cyclic AMP levels in intact prelabeled cells. The ATP pool was prelabeled by incubating the cells in the presence of radioactive adenine. The cyclic AMP formed from ATP was extracted with HC10(4) and separated from adenine and other adenosine-related nucleotides by chromatography on acriflavin-Sephadex G-25. This method provides a rapid and sensitive isolation of cyclic AMP with high recovery (95-100%) and low blnks. Further, no contamination of the cyclic AMP fractions was found by either adenine or adenosine nucleotides such as ATP, ADP or AMP. This procedure is applicable to a variety of cell or tissue systems.     

Modifications in the allosteric properties of phosphofructokinase in rat erythrocytes and reticulocytes cross-linked with dimethyl suberimidate and 3,3'-dithiobispropionimidate

The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U     

Biosynthesis of heme in immature erythroid cells. The regulatory step for heme formation in the human erythron

Heme formation in reticulocytes from rabbits and rodents is subject to end product negative feedback regulation: intracellular "free" heme has been shown to control acquisition of transferrin iron for heme synthesis. To identify the site of control of heme biosynthesis in the human erythron, immature erythroid cells were obtained from peripheral blood and aspirated bone marrow. After incubation with human 59Fe transferrin, 2-[14C]glycine, or 4-[14C]delta-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glycine and iron but not delta-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 microM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular 59Fe into heme by human erythroid cells but assimilation of 4-[14C]delta-aminolevulinate into heme was insensitive to inhibition by hematin (Ki greater than 100 microM). In human reticulocytes (unlike those from rabbits), addition of ferric salicylaldehyde isonicotinoylhydrazone, to increase the pre-heme iron pool independently of the transferrin cycle, failed to promote heme synthesis or modify feedback inhibition induced by hematin. In human erythroid cells (but not rabbit reticulocytes) pre-incubation with unlabeled delta-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell 59Fe for heme synthesis and also attenuated end product inhibition. In human erythroid cells heme biosynthesis is thus primarily regulated by feedback inhibition at one or more steps which lead to delta-aminolevulinate formation. Hence in man the regulatory process affects generation of the first committed precursor of porphyrin biosynthesis by delta-aminolevulinate synthetase, whereas in the rabbit separate regulatory mechanisms exist which control the incorporation of iron into protoporphyrin IX.     

Inhibition of peptide chain initiation in lysates from ATP-depleted cells. II. Studies on the mechanism of the lesion and its relation to similar alterations caused by oxidized glutathione and hemin deprivation.

The impairment of peptide chain initiation in lysates from ATP-depleted rabbit reticulocytes is accompanied by a loss of their ability to form the 40 S methionyl-tRNAfMet complex with a resultant failure to promote the AUG codon-dependent combination of the complex with the 60 S ribosomal subunit. These partial initiation reactions, as well as the overall protein-synthetic activity of the defective lysates could be restored by addition of a 0.5 M KC1 ribosomal extract or normal postribosomal supernatant or a 40-70% (NH4)2-SO4 fraction derived from it. Alternatively, reactivation of the impaired lysates could be achieved by supplementation with millimolar amounts of cyclic AMP or certain purine derivatives. The same subcellular fractions, as well as cyclic AMP or purine derivatives were also capable of overcoming the inhibition caused by incubating reticulocyte lysates in the presence of oxidized glutathione or in the absence of hemin. Severe intracellular ATP deprivation resulted in accumulation of a soluble translational inhibitor in the postribosomal fraction, thus resembling the parallel phenomenon described in hemin-deprived lysates. The striking similarities between the three kinds of inhibition studied by us point to an identical site of the underlying biochemical lesion, despite the different mechanisms mediating their induction.     

Role of AMP on the activation of glycogen synthase and phosphorylase by adenosine, fructose, and glutamine in rat hepatocytes

The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.     

Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP

NADH oxidase activity of rat liver plasma membranes was inhibited by lowconcentrations (1-100 nM) of ATP. The inhibition was amplified by additionof nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition wascomplex and related to a marked increase in the Km for NADH at high NADHconcentrations together with a concomitant decrease in the Vmax. In theabsence of added or residual ATP, cyclic AMP was without effect. Theresponse of cyclic AMP + ATP was inhibited by low concentrations of theselective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not bystaurosporin. The Vmax but not the Km was modified by treating the plasmamembranes with a mild oxidizing agent, N-chlorosuccinamide, or with thereducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmaxwas reduced by cyclic AMP + ATP. In contrast, in the presence ofN-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative tocyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmaxcould be either an increase or a decrease depending on whether the membraneswere oxidized or reduced. The results demonstrate regulation of NADH oxidaseactivity of rat liver plasma membranes through cyclic AMP-mediatedphosphorylation by membrane-located protein kinase activities where thefinal response is dependent on the oxidation-reduction status of the plasmamembranes.     

Measurement of cyclic 3',5'-adenosine monophosphate synthesis in growing Escherichia coli.

The net synthesis of cAMP by an adenine auxotroph of $\text{Escherichia coli}$ was measured by assaying the incorporation of tritium from [³H]-adenine into cyclic [³H] AMP during exponential growth. Synthesis of cAMP ceased abruptly when glucose was added to cells growing in glycerol and then recovered to an intermediate rate of synthesis after 0.5–1.0 generation. Cyclic AMP appeared to be synthesized from a precursor pool that turned over more rapidly than total cellular ATP. The rates of cAMP synthesis measured by this technique are compatible with the cellular levels of cAMP previously measured in this strain(3).     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Adenine nucleotide metabolism in isolated chicken hepatocytes.

The turnover of the adenine nucleotide pool, the pathway of the degradation of AMP and the occurrence of recycling of adenosine were investigated in isolated chicken hepatocytes, in which the adenylates had been labelled by prior incubation with [14C]adenine. Under physiological conditions, 85% of the IMP synthesized by the 'de novo' pathway (approx. 37 nmol/min per g of cells) was catabolized directly via inosine into uric acid, and 14% was converted into adenine nucleotides. The latter were found to turn over at the rate of approx. 5 nmol/min per g of tissue. Inhibition of adenosine deaminase by 1 microM-coformycin had no effect on the formation of labelled uric acid, indicating that the initial degradation of AMP proceeds by way of deamination rather than dephosphorylation. Inhibition of adenosine kinase by 100 microM-5-iodotubercidin resulted in a loss of labelled ATP, demonstrating that adenosine is normally formed from AMP but is recycled. Unexpectedly, 5-iodotubercidin did not decrease the total concentration of ATP, indicating that the loss of adenylates caused by inhibition of adenosine kinase was nearly completely compensated by formation of AMP de novo. Anoxia induced a greatly increased catabolism of the adenine nucleotide pool, which proceeded in part by dephosphorylation of AMP. On reoxygenation, the formation of AMP de novo was increased 8-fold as compared with normoxic conditions. The latter results indicate the existence of adaptive mechanisms in chick liver allowing, when required, channelling of the metabolic flux through the 'de novo' pathway, away from the uricotelic catabolic route, into the synthesis of adenine nucleotides.     

In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli

The incorporation of [14C]adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with 60Co gamma-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of gamma-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after higher doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m-2) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as gamma-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.     

Role of fructose 2,6-bisphosphate in the stimulation of glycolysis by anoxia in isolated hepatocytes.

1. Incubation of hepatocytes from fed or starved rats with increasing glucose concentrations caused a stimulation of lactate production, which was further increased under anaerobic conditions. 2. When glycolysis was stimulated by anoxia, [fructose 2,6-bis-phosphate] was decreased, indicating that this ester could not be responsible for the onset of anaerobic glycolysis. In addition, the effect of glucose in increasing [fructose 2,6-bisphosphate] under aerobic conditions was greatly impaired in anoxic hepatocytes. [Fructose 2,6-bisphosphate] was also diminished in ischaemic liver, skeletal muscle and heart. 3. The following changes in metabolite concentration were observed in anaerobic hepatocytes: AMP, ADP, lactate and L-glycerol 3-phosphate were increased; ATP, citrate and pyruvate were decreased: phosphoenolpyruvate and hexose 6-phosphates were little affected. Concentrations of adenine nucleotides were, however, little changed by anoxia when hepatocytes from fed rats were incubated with 50 mM-glucose. 4. The activity of ATP:fructose 6-phosphate 2-phosphotransferase was not affected by anoxia but decreased by cyclic AMP. 5. The role of fructose 2,6-bisphosphate in the regulation of glycolysis is discussed.     

A simple assay for adenylate cyclase in intact cells by affinity elution chromatography.

A method using the principle of affinity elution chromatography is described for the assay of adenylate cyclase in intact human platelets. By incubating platelet-rich plasma in the presence of radioactively labelled adenine, the ATP pool of the cells was prelabelled. Formation of labelled cyclic AMP from ATP was determined by extracting the platelets with HC1O4. After removal of the latter as KC1O4, the extract containing cyclic AMP and other adenine nucleotides was adsorbed in a NN-diethyl-N-2-hydroxypropylamino (QAE)-cellulose column. The column was washed, and subsequently cyclic AMP was specifically eluted with a cyclic AMP-dependent protein kinase and the radioactivity of the eluate was determined.     

Studies on rat liver catalase. X. Effect of hemin and an inhibitor on the translation of catalase messenger RNA1.

Rat liver catalase mRNA was translated in a rabbit reticulocyte lysates and wheat germ cell-free system in the presence or absence of hemin and/or a translational inhibitor prepared from reticulocytes, liver cells, and wheat germs. Failure to add hemin to the lysates, or the addition of a hemin-regulated translational inhibitor (HRI) to the hemin-supplemented lysates caused a repressed translation. A preparation of inhibitor from rat liver showed activity similar to that of HRI for this translating system. The translation repression by rat liver inhibitor was reversed by eIF-2 (initiation factor) or GTP, but ATP enhanced the repression. The translation of catalase mRNA in the wheat germ system was not affected by the addition of hemin. An inhibitor prepared from wheat germ extracts, as well as the rat liver inhibitor, markedly decreased the rate of translation. eIF-2, GTP, and ATP behaved in the manner described above. Catalase synthesis in a cell-free system derived from rat liver (using endogenous mRNA) was not influenced by either hemin or the inhibitor. The possibilities are discussed that the synthesis of catalase in liver cells is controlled by a translational inhibitor at the level of chain initiation, and that the formation of the inhibitor from its inactive proinhibitor is regulated by the amount of heme.     

Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.     

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.     

Elevation of the adenylate pool in rat cardiomyocytes by S-adenosyl-L-methionine

Rapid resynthesis of the adenylate pool in cardiac myocytes is important for recovery of contractility and normal function of regulatory mechanisms in the heart. Adenosine and adenine are thought to be the most effective substrates for nucleotide synthesis, but the possibility of using other compounds has been studied very little in cardiomyocytes. In the present study, the effect of S-adenosyl-L-methionine (SAM) on the adenylate pool of isolated cardiomyocytes was investigated and compared to the effect of adenine and adenosine. Adult rat cardiomyocytes were isolated using the collagenase perfusion technique. The cells were incubated in the presence of adenine derivatives for 90 min followed by nucleotide determination by HPLC. The concentrations of adenine nucleotides expressed in nmol/mg of cell protein were initially 22.1 +/- 1.4, 4.0 +/- 0.3 and 0.70 +/- 0.08 for ATP, ADP and AMP, respectively (n = 10, +/- S.E.M.), and the total adenylate pool was 26.8 +/- 1.6. In the presence of 1.25 mM SAM in the medium, the adenylate pool increased by 5.2 +/- 0.4 nmol/mg of cell protein, but only if 1 mM ribose was additionally present in the medium. No changes were observed with SAM alone. A similar increase (by 4.9 +/- 0.6 nmol/mg protein) was observed after incubation with 1.25 mM adenine plus 1 mM ribose, but no increase was observed if ribose was omitted. Adenosine at 0.1 or 1.25 mM concentrations also caused an increase in the adenylate pool (by 5.2 +/- 1.0 and 5.2 +/- 0.9 nmol/mg protein, respectively), which in contrast to the SAM or adenine was independent of the additional presence of ribose. Thus, S-adenosyl-L-methionine could be used as a precursor of the adenylate pool in cardiomyocytes, which is as efficient in increasing the adenylate pool after 90 min of incubation as adenosine or adenine. Nucleotide synthesis from SAM involves the formation of adenine as an intermediate with its subsequent incorporation by adenine phosphoribosyltransferase.     

Effects of nitroglycerin on energy metabolism of rat reticulocytes.

Nitric oxide (NO) in many cells inactivates aconitase and mitochondrial respiratory chain, and influenced glyceraldehyde 3-phosphate dehydrogenase activity. The aim of this study was to evaluate role of nitroglycerin (NTG), a widely used NO donor, on energy metabolism of rat reticulocytes. Rat reticulocyte rich red blood cell suspensions containing 70-100% of reticulocytes, were aerobically incubated without (control) or in the presence of different concentrations of (a) NTG (0.1, 0.25, 0.5, 1.0, 1.5 mmol/l), (b) 8-Br-cGMP (0.1, 0.5, 1.0 mmol/l) and (c) NaNO2 and NaNO3 (1 mmol/l). NTG in dose- and time-dependent manner decreased total (p>0.05; EC50 = 0.78+/-0.05 mmol/l) and coupled (p<0.05; EC50 = 0.50+/-0.04 mmol/l) and increased uncoupled oxygen consumption (p<0.05: EC50 = 0.36+/-0.01 mmol/l). They were accompanied by stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.001 EC50 = 0.53 and 0.53 mmol/l, respectively). Levels of all glycolytic intermediates in the presence of NTG indicate stimulation of HK-PFK, GA3PDH and PK activity. NTG significantly decreased ATP level, which accompanied by increased ADP and AMP levels. However, level of total adenine nucleotides (TAN) was significantly lower, which was consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level; p<0.05). Stimulation of glycolysis accompanied with inhibition of the OxP, activation of HK-PFK, decrease of ATP and simultaneous rise of ADP and AMP levels, all together represent an example of Pasteur effect occurring in NTG-treated reticulocytes. In rat reticulocytes under steady state conditions 93% of overall energy was produced by OxP, but only 7% by glycolysis. Due to decrease of coupled oxygen consumption in the presence of NTG, ATP production via OxP was significantly diminished. Simultaneous increase of glycolytic ATP production is not enough to provide constant either ATP production or concentration. Calculated mean ATP-turnover time was prolonged even for 45% in the presence of 1.5 mmol/l NTG. Metabolic effects of NTG were not mimic by exogenous 8-Br-cGMP, NaNO2 or NaNO3, which indicate that NTG induced a) inhibition of coupled respiration and b) stimulation of glycolysis in rat reticulocytes are mediated by NO as an effector molecule.     

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.     

Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity

The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc in granulosa cells.