Anti-anabolic effects of adenosine 3':5'-cyclic monophosphate. Inhibition of protein synthesis.
ABSTRACT: 1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.
Project description:1. Gluconeogenesis from pyruvate was measured in renal-cortical-tubules fragments prepared from fed male rats 6-8 days after adrenalectomy or sham adrenalectomy. The response of this process to 3':5'-cyclic AMP and adrenaline was compared in these two states at two Ca2+ concentrations. 2. Adrenalectomy decreased the percentage stimulation of gluconeogenesis by 3':5'-cyclic AMP, but increased percentage stimulation by adrenaline. Cortisol treatment of adrenalectomized rats (50 mg/kg, twice daily for 2 days) did not reverse the change in responsiveness to 3':5'-cyclic AMP and adrenaline. 3. Stimulation of gluconeogenesis by 1 micron-adrenaline was unaffected by 10 micron-propranolol (beta-blocker) in either state. Phentolamine (alpha-blocker; 10 micron) totally blocked stimulation of gluconeogenesis by 1 micron-adrenaline in the sham-operated condition, but was only partially effective in this respect after adrenalectomy.
Project description:Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 126.96.36.199) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.
Project description:1. By perfusion of rat livers with 3mm-AMP in the perfusion medium we obtain increased intracellular concentrations of AMP. 2. These high intracellular concentrations of AMP lead to an increased output of glucose and urea into the perfusion medium. 3. The increased output of glucose in livers from fed rats is brought about primarily by an AMP-stimulated breakdown of liver glycogen. In livers from starved rats the increase in glucose output is not as great, reflecting the low contents of glycogen in livers from starved rats. 4. AMP inhibits gluconeogenesis from lactate in perfused livers. In the presence of high concentrations of lactate, however, the counteracting effects of AMP to increase glycogenolysis and to inhibit gluconeogenesis result in little change in the net glucose output. 5. The increased urea output is brought about by increased breakdown of amino acids that are present in the perfusion medium. In livers from starved rats the overall urea production is much higher, indicating increased catabolism of amino acids and other nitrogenous substrates in the absence of carbohydrate substrates. 6. AMP causes an inhibition of incorporation of labelled precursors into protein and nucleic acid. This may result from increased catabolism of precursors of proteins and nucleic acids as reflected by the more rapid breakdown of nitrogenous compounds. In support of this hypothesis, cell-free systems for amino acid incorporation isolated from livers perfused with and without AMP are equally capable of supporting protein synthesis. 7. The labelling pattern of RNA in perfused livers corresponds very closely to those found by pulse-labelling in vivo. AMP in no way alters the qualitative nature of the labelling patterns. 8. We consider these results as supporting evidence for the role of the concentration ratio of AMP to ATP in controlling the metabolic pathways that lead to the formation of ATP.
Project description:Free ribosomes and a smooth-microsomal fraction were prepared from bovine corpus luteum. Both preparations will self-phosphorylate when incubated with Mg(2+) and ATP, but at low concentrations of Mg(2+) and ATP the self-phosphorylation of the smooth-microsomal fraction was much more dependent on cyclic AMP than was that of free ribosomes, stimulation by the nucleotide being up to 10-fold in the former case. The self-phosphorylation of the smooth-microsomal fraction was studied further. The reaction bears similarities to that brought about by soluble cyclic AMP-dependent protein kinase, being inhibited by Ca(2+) and the heat-stable inhibitor protein from skeletal muscle. Cyclic GMP will activate the reaction at concentrations higher than those required for full activation by cyclic AMP. In the presence of cyclic AMP, phosphate bound to protein is found almost exclusively as phosphoserine. Several proteins are phosphorylated, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the phosphorylation of all of them is markedly stimulated by cyclic AMP. If the reaction is carried out at high concentrations of Mg(2+) and ATP, a distinct cyclic AMP-independent phosphorylation is observed. This activity is not inhibited by the heat-stable inhibitor protein, and phosphate is found esterified with both threonine and serine residues.
Project description:The equilibrium binding of cyclic AMP to a 150-fold purified preparation of protein kinase, when expressed as the reciprocal of bound against the reciprocal of free cyclic AMP, gave a plot consisting of two straight lines. The values of apparent Kb given by these lines were lowered by preincubating the intact tissue with noradrenaline or incubating the enzyme preparation with Mg2+ plus ATP. This effect was reversed by incubating the preparation (which contained some phosphatase impurities) with Mg2+ alone. None of these procedures affected the maximal binding of cyclic AMP. During incubation of the enzyme with Mg2+ plus ATP, the terminal phosphoryl group was incorporated into protein, over 40% being present in the kinase itself. This phosphate was removed during incubation of the preparation with Mg2+ alone. The validity of expressing cyclic AMP binding as a double-reciprocal plot is discussed, and the experimental plots are compared with those derived theoretically. The results suggest that protein kinase in brown fat is present in two forms, one with an apparent Kb for cyclic AMP or approx. 250 nM (dephosphorylation) and one with an apparent Kb of approx. 14 nM (phosphorylated). Preincubation of the tissue with noradrenaline results in phosphorylation of the kinase and an increase from 15 to 45% in the proportion of the higher-affinity form.
Project description:Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 188.8.131.52) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as the cyclic AMP concentration (Ka) at which the protein kinase had 50% of its maximal activity. The close correlation between binding and activation is also found in the presence of KCl, which increased the apparent activation constant (Ka) for cyclic AMP. With increasing [KCl], a progressively higher proportion of the histone phosphorylation observed in cytosol was due to cyclic AMP-independent (casein) kinases, leading to an overestimation of the degree of activation of the cyclic AMP-dependent protein kinases present. The relative contributions of cyclic AMP-dependent and -independent kinases to histone phosphorylation at different ionic strengths was determined by use of heat-stable inhibitor and phospho-cellulose chromatography.
Project description:Glycerol release and tissue concentrations of ATP and cyclic AMP were followed during the incubation of adipose tissue with or without glucose, insulin and noradrenaline. Glucose plus insulin or, to a lesser extent, glucose alone increased the accumulation of glycerol during incubations both with and without noradrenaline by slowing the decline in the rate of glycerol release with time. Insulin alone decreased the accumulation by accelerating the fall in glycerol release. In the absence of noradrenaline, ATP and cyclic AMP concentrations were not significantly affected by insulin or glucose. With noradrenaline or noradrenaline plus insulin the ATP concentration gradually fell. With noradrenaline plus glucose the ATP concentration fell rapidly and then stabilized, or, if insulin was also present, returned to the control value. In the presence of noradrenaline, the concentration of cyclic AMP rose during the first 20min and then fell. Insulin lowered the peak concentration of cyclic AMP, but glucose had no effect either on the peak value or the fall in the concentration of the nucleotide. The increase and fall in the concentration of cyclic AMP with noradrenaline or noradrenaline plus insulin bore similarities to the increase and decline in the lipolytic rate in incubations without glucose. It is proposed that glucose stimulates ATP production by furnishing glycerol 1-phosphate and thus removing free fatty acids, but that it can influence lipolysis by a mechanism which is distinct from any which is mediated by free fatty acids, possibly by inhibiting the inactivation of the lipase.
Project description:1. Sealed pigeon erythrocyte 'ghosts' were prepared containing ATP and the Ca2+-activated photoprotein obelin to investigate the relationship cyclic AMP formation and internal free Ca2+. 2. The 'ghosts' were characterized by (a) morphology (optical and electron microscopy), (b) composition (haemoglobin, K+, Na+, Mg2+, ATP, obelin), (c) permeability to Ca2+, assessed by obelin luminescence and (d) hormone sensitivity (the effect of beta-adrenergic agonists and antagonists on cyclic AMP formation). 3. The effect of osmolarity at haemolysis and ATP at resealing on these parameters was investigated. 4. Sealed 'ghosts', containing approx. 2% of original haemoglobin, 150mM-K+, 0.5MM-ATP, 10(3)--10(4) obelin luminescence counts/10(6) 'ghosts', which were relatively impermeable to Ca2+ and in which cyclic AMP formation was stimulated by beta-adrenergic agonists over a concentration range similar to that for intact cells, could be prepared after haemolysis in 6mM-NaCl3mM-MgCl2/50mM-Tes, pH7, and resealing for 30min at 37 degrees C in the presence of ATP and 150mM-KCl. 5. The initial rate of adrenaline-stimulated cyclic AMP formation in these 'ghosts' was 30--50% of that in intact cells and was inhibited by the addition of extracellular Ca2+. Addition of Ca2+ to the 'ghosts' resulted in a stimulation of obelin luminescence, indicating an increase in internal free Ca2+ under these conditions. 6. The ionophore A23187 increased the rate of obelin luminescence in the 'ghosts' and also inhibited the adrenaline-stimulated increase in cyclic AMP. 7. The effect of ionophore A23187 on obelin luminescence and on cyclic AMP formation in the 'ghosts' was markedly decreased by sealing EGTA inside the 'ghosts'. 8. It was concluded that cyclic AMP formation inside sealed pigeon erythrocyte 'ghosts' could be inhibited by more than 50% by free Ca2+ concentrations in the range 1--10 micrometer.
Project description:Cyclic AMP causes an age-dependent inhibition of protein synthesis in rat liver. The onset of inhibition is about 10--12 days after birth. The developmental response to cyclic AMP is associated with a change in the microsomal component of the protein-synthesizing system.
Project description:1.3':5'-Cyclic AMP was extensively purified from Kalanchoe daigremontiana and Agave americana by neutral alumina and anion- and cation-exchange column chromatography. Inclusion of 3':5'-cyclic [8-3H]AMP from the point of tissue extraction permitted calculation of yields. The purification procedure removed contaminating material that was shown to interfere with the 3':5'-cyclic AMP estimation and characterization procedures. 2. The partially purified 3':5'-cyclic AMP was quantified by means of a radiochemical saturation assay using an ox heart 3':5'-cyclic AMP-binding protein and by an assay involving activation of a mammalian protein kinase. 3. The plant 3':5'-cyclic AMP co-migrated with 3':5'-cyclic [8-3H]AMP on cellulose chromatography, poly(ethyleneimine)-cellulose chromatography and silica-gel t.l.c. developed with several solvent systems. 4. The plant 3':5'-cyclic AMP was degraded by ox heart 3':5'-cyclic nucleotide phosphodiesterase at the same rates as authentic 3':5'-cyclic AMP. 1-Methyl-3-isobutylxanthine (1 mM), a specific inhibitor of the 3':5'-cyclic nucleotide phosphodieterase, completely inhibited such degradation. 5. The concentrations of 3':5'-cyclic AMP satisfying the above criteria in Kalanchoe and Agave were 2-6 and 1 pmol/g fresh wt. respectively. Possible bacterial contribution to these analyses was estimated to be less than 0.002pmol/g fresh wt. Evidence for the occurrence of 3':5'-cyclic AMP in plants is discussed.