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PubMed2003

Modulation of the reactive oxygen species (ROS) generation mediated by cyclic AMP-elevating agents or Interleukin 10 in granulocytes from type 2 diabetic patients (NIDDM): a PKA-independent phenomenon.

UNLABELLED: SUMMARY-BACKGROUND: The present study investigates the hypothesis that cells from ill patients and from healthy subjects may have different reactivity under metabolic stimulation as a consequence of an disease-induced metabolic adaptation. METHODS: Granulocytes either from healthy subjects or from type II-Non Insulin Dependent Diabetes Mellitus (NIDDM) patients were compared in their capacities to generate Reactive Oxygen Species (ROS). The ROS generation was comparatively determined in a chemiluminescence assay, luminol-dependent, after cell incubation in the presence of either cyclic AMP - elevating agents or Interleukin 10. In some experiments the cells were pretreated with H89 compound (a PKA inhibitor) or with diphenylene iodonium (DPI), a NADPH-oxidase inhibitor. RESULTS: Our results showed an increased ROS generation in granulocytes from diabetic patients in absence of cyclic AMP-elevating agents or IL-10. In the presence of cyclic AMP-elevating agents was observed an inverse metabolic response in granulocytes from diabetic patients in comparison to cells from healthy subjects. The granulocytes were pre-incubated in the presence of cyclic AMP-elevating agents--amminophylline (AMF) or dibutyryl cyclic AMP (dbcAMP)--or interleukin 10 (IL-10). The AMF, dbcAMP and IL-10 inhibited ROS production by granulocytes from healthy subjects. By contrast, AMF and dbcAMP activated cells from diabetic patients while IL-10 had no effect. The inhibition of ROS induced by AMF, dbcAMP or IL-10 was promptly abolished by the pretreatment of the cells with either PKA H89 inhibitor or NADPH-oxidase inhibitor (DPI) in granulocytes from healthy subjects. In relation to the granulocytes from type 2 diabetics patients, the activation of ROS generation mediated by AMF and dbcAMP was fully abolished by NADPH-oxidase DPI-inhibitor, but not by PKA H89 inhibitor. CONCLUSIONS: Our present results reinforce the hypothesis that cells from ill patients (type II diabetic) when compared to cells from healthy subjects have different reactivity under metabolic stimulation. ROS production by human granulocytes was modulated by cyclic AMP elevating agents and IL-10. The inhibition of the ROS production in cells from healthy subjects was PKA-dependent while the activation in granulocytes from patients was PKA-independent. This inverse metabolic response, in cells from patients, suggests the use of an alternative metabolic pathway PKA-independent, possible cAMP/Epac/PKB-dependent. The correlation between activation of ROS production in granulocytes from diabetic patients and pathogenesis of diabetes can be suggested, however, further and extensive studies are needed for demonstrating this suggestion.

Aged↗
PubMed1998

Continuous activation of pituitary adenylate cyclase-activating polypeptide receptors elicits antipodal effects on cyclic AMP and inositol phospholipid signaling pathways in CATH.a cells: role of protein synthesis and protein kinases.

Continuous exposure of cells to agonists develops a process that determines the extent to which the cells eventually respond to further stimuli. Here we used CATH.a cells (a catecholaminergic neuron-like cell line), which express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors linked to both adenylyl cyclase and phospholipase C-beta pathways, to investigate the influence of prolonged hormonal treatment on dual signaling and gene transcription. Prolonged incubation of cells with PACAP failed to down-regulate the density and affinity of membrane binding sites and caused opposite changes in messenger systems: PACAP-stimulated cyclic AMP accumulation was attenuated in a time- and dose-dependent fashion (t(1/2) = 6.7 h and IC50 = 0.1 nM), whereas phosphoinositide turnover was overstimulated. Both effects were insensitive to pertussis toxin, whereas the drop in cyclic AMP concentration was also unchanged in the presence of 3-isobutyl-1-methylxanthine, indicating that neither Gi-like proteins nor cyclic nucleotide phosphodiesterases play a critical role in these processes. Blockade of protein synthesis with cycloheximide, as well as inhibition by H89 of cyclic AMP-dependent protein kinase (but not by bisindolylmaleimide of protein kinase C) antagonized the influences exerted by PACAP on adenylyl cyclase activity and inositol phosphate formation. Transcription of the chimeric GAL4-CREB construct, transiently transfected into CATH.a cells, was stimulated by PACAP, and this effect was potentiated as a result of chronic PACAP treatment. The results of the present investigation provide new insight into the possible differential regulation and cross-talks of transduction signals of receptors linked to multiplex signaling. They demonstrate that prolonged exposure of CATH.a cells to PACAP results in the desensitization of the cyclic AMP pathway and superinduction of the inositol phosphate signal, through protein neosynthesis and cyclic AMP-dependent protein kinase activation. At the same time, they show that desensitization of cyclic AMP signaling not only fails to hamper, but actually amplifies PACAP-stimulated CREB-regulated transcription.

Cell Line↗
PubMed1987

Dibutyryl cyclic AMP inhibits transport dependent QO2 in cells isolated from the rabbit medullary ascending limb.

Because the medullary thick ascending limb of the loop of Henle is the target of several polypeptide hormones that stimulate adenyl cyclase in this nephron segment, we examined the effects of cyclic AMP on thick ascending limb of the loop of Henle cells isolated by enzymatic digestion and density gradient centrifugation from the outer medulla of the rabbit kidney. The functional parameter that was measured was transport dependent oxygen consumption. Oxygen consumption was measured using a polarographic oxygen electrode in a constant temperature chamber. We found that dibutyryl cyclic AMP inhibited oxygen consumption in a dose dependent way. Maximal inhibition was observed at a concentration of 10(-5) M. The effect of dibutyryl cyclic AMP was not present in the absence of either sodium, chloride or both implying that its effect is restricted to the sodium and chloride dependent oxygen consumption. The effect of dibutyryl cyclic AMP was additive to that of furosemide 10(-4) M while that of furosemide was not additive to that of cyclic AMP suggesting that the site of action of cyclic AMP is distal to that of furosemide. The effect of dibutyryl cyclic AMP was not additive to that of ouabain and was absent in cells where oxygen consumption was stimulated with amphotericin B in the absence of chloride indicating that it has no effect on Na-K-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Divalent cations modify adsorption of 5'-AMP onto precipitated calcium phosphate: a model for cation modulation of adsorptive processes in primitive aqueous environments.

The adsorption of 5'-AMP onto precipitated calcium phosphate (CaPi) requires the presence of soluble calcium and this dependence exhibits a Michaelian-like behavior. This result suggests that the formation of a complex between 5'-AMP and free Ca2+ (CaAMP) is a prelude to the adsorption of the nucleotide in the solid matrix. At concentrations one order of magnitude higher, Mn2+ and Mg2+ can substitute for soluble Ca2+ in the adsorption of 5'-AMP onto solid CaPi. However, when added simultaneously with 5'-AMP to a heterogeneous mixture that contains CaPi and soluble Ca2+, Mn2+ and Mg2+ inhibit the adsorption of 5'-AMP in a concentration-dependent manner. This suggests the formation of complexes that are much less effective for 5'-AMP adsorption than the CaAMP complex. On the other hand, Mn2+ and Mg2+ cannot promote desorption of the nucleotide attached to the precipitate in the presence of soluble Ca2+ if they are added after adsorption has attained equilibrium. Although desorption of 5'-AMP can be obtained by a sequential dilution of the soluble phase with buffer and no nucleotide in a process that obeys a Langmuir equation, the lack of effect of Mn2+ or Mg2+ when adsorption has attained its maximal value suggests strong interactions between the CaAMP complex and the solid matrix when adsorption equilibrium is reached. The divalent cations present in the matrix also participate with different selectivity in the attachment of the CaAMP complex, indicating that a cation-exchange mechanism could have acted in the modulation of adsorptive/desorptive processes involving biomonomers and phosphate surfaces in primitive aqueous environments.

Adenosine Monophosphate↗
PubMed1986

Cyclic AMP and Ca2+ uptake by mastocytoma mitochondria.

A thorough re-investigation was undertaken of a variety of factors that might explain the increased uptake of 45Ca2+ by mitochondria isolated from N6, O2'-dibutyryladenosine-3',5'-cyclic monophosphate (DB cyclic AMP)--treated PY815 cells. This showed that mitochondria isolated from DB cyclic AMP treated cells take up 45Ca2+ at a 30 per cent faster rate than mitochondria from untreated cells, although both mitochondria eventually reduce the total external Ca2+ to the same levels. 45Ca2+ precharged mitochondria from DB cyclic AMP-treated cells also leaked 45Ca2+ more slowly than those from untreated cells when they were recovered by filtration. Thus an apparently greater uptake of 45Ca2+ by mitochondria from DB cyclic AMP-treated cells was a consequence of the filtration procedure. In fact, mitochondria from DB cyclic AMP-treated cells contained less total Ca2+ than those from untreated cells, while DB cyclic AMP-treated cells also contained less total Ca2+ than untreated cells. The results suggest that mitochondria do not play an important role in controlling the growth of DB cyclic AMP-treated PY815 cells through effects on cytoplasmic Ca2+ availability.

Adenosine Triphosphate↗
PubMed1986

Cyclic AMP-dependent phosphorylation of high molecular mass proteins in pig thyroid cells.

Four high molecular mass (H Mr) proteins were found to be phosphorylated in a cyclic AMP-dependent manner in both partially purified pig thyroid membrane fractions and in pig thyroid cells in culture. These phosphoproteins did not correspond to major cellular proteins; they were found in both soluble and particulate subfractions of homogenates from cultured thyroid cells. The molecular mass of the 4 proteins named HMr1 to HMr4 determined by polyacrylamide gel electrophoresis in the in the presence of sodium dodecylsulfate was about 310 000 for HMr1, 250 000 for HMr2, 240 000 for Hmr3 and 220 000 for HMr4. HMr1 comigrated with brain MAP1, whereas HMr3 and HMr4 had the same mobility as alpha-and beta-spectrins, respectively. The 4 high molecular mass phosphoproteins are substrates of cyclic AMP-dependent protein kinase(s) since (a) their phosphorylation was increased by cyclic AMP and not by cyclic GMP or calcium alone or calcium in the presence of calmodulin or phospholipid; (b) the effect of cyclic AMP was prevented by the thermostable inhibitor of cyclic AMP-dependent protein kinases; (c) the purified catalytic subunit of cyclic AMP-dependent protein kinases markedly phosphorylated the 4 HMr proteins. The 32P-labeling of HMr proteins using either endogenous cyclic AMP-dependent protein kinase or the purified catalytic subunit was always lower in cells cultured in the presence of TSH (reassociated in follicle-like structures) than in freshly dispersed cells or cells cultured in basal conditions (cells in monolayer). These results suggest that the 4 high molecular mass thyroid phosphoproteins represent structural components, the phosphorylation of which could vary with the cellular organization.

Animals↗
PubMed2005

The origin of the high sensitivity of muscle fructose 1,6-bisphosphatase towards AMP.

Adenosine 5'-monophosphate (AMP) inhibits muscle fructose 1,6-bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild-type muscle enzyme, but it binds the fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), similarly to the wild-type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP-AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission.

Adenosine Monophosphate↗
PubMed2003

TNP-AMP binding to the sarcoplasmic reticulum Ca(2+)-ATPase studied by infrared spectroscopy.

Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.

Adenosine Monophosphate↗
PubMed1999

Increased hypoxic stress decreases AMP hydrolysis in rabbit heart.

OBJECTIVE: AMP conversion to adenosine by cytosolic 5'nucleotidase (5NT) or to IMP by AMP deaminase determines the degree of nucleotide degradation, and thus ATP resynthesis, during reoxygenation. To elucidate the regulation of AMP hydrolysis during ischemia, data from 31P NMR spectroscopy and biochemical analyses were integrated via a mathematical model. Since 5NT is downregulated during severe underperfusion (5% flow), we tested 5NT regulation during less severe underperfusion (10% flow) and then made the perfusate hypoxic to see if the greater stress reactivated 5NT. METHODS: 31P NMR spectra and coronary venous effluents were obtained from Langendorff-perfused rabbit hearts subjected to two 30-min periods of underperfusion (10% flow); the second period with or without additional hypoxia (30% O2). Data were analyzed with a mathematical model describing the kinetics of myocardial energetics and metabolism. RESULTS: A single 30-min period of 10% flow causes downregulation of AMP hydrolysis and the data from the second period of underperfusion are best described by lower 5NT activity, even in the presence of extra hypoxia. Thirty percent less purines appear in the venous effluent than predicted by the phosphoenergetics (PCr and ATP) when IMP is not allowed to accumulate by the model, however the model indicates that a constant accumulation of IMP via AMP deaminase could explain the discrepancy between expected and measured purines in the venous effluent. CONCLUSIONS: While AMP hydrolysis to adenosine is prominent in early ischemia and acts to preserve cellular energy potential, during a second ischemic period, nucleotides are conserved by the stable inhibition of AMP hydrolysis. Furthermore, during 10% flow conditions, nucleotides are conserved, possibly via an IMP-accumulatory pathway.

Adenosine↗
PubMed2001

AMP inhibition of pig kidney fructose-1,6-bisphosphatase.

Lys-112 and Tyr-113 in pig kidney fructose-1,6-bisphosphatase (FBPase) make direct interactions with AMP in the allosteric binding site. Both residues interact with the phosphate moiety of AMP while Tyr-113 also interacts with the 3'-hydroxyl of the ribose ring. The role of these two residues in AMP binding and allosteric inhibition was investigated. Site-specific mutagenesis was used to convert Lys-112 to glutamine (K112Q) and Tyr-113 to phenylalanine (Y113F). These amino acid substitutions result in small alterations in k(cat) and increases in K(m). However, both the K112Q and Y113F enzymes show alterations in Mg(2+) affinity and dramatic reductions in AMP affinity. For both mutant enzymes, the AMP concentration required to reduced the enzyme activity by one-half, [AMP](0.5), was increased more than a 1000-fold as compared to the wild-type enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg(2+). Although the allosteric site is approximately 28 A from the metal binding sites, which comprise part of the active site, these site-specific mutations in the AMP site influence metal binding and suggest a direct connection between the allosteric and the active sites.

Adenosine Monophosphate↗
PubMed1981

Periodate-oxidized AMP as a substrate, an inhibitor and an affinity label of human placental alkaline phosphatase.

Human placental alkaline phosphatase (EC 3.1.3.1) was inactivated by periodate-oxidized AMP. The inactivation showed saturation kinetics and could be partially prevented by the substrate AMP or the product inhibitor inorganic phosphate. Oxidized AMP was itself a substrate for this enzyme, with an apparent Km of 0.67 mM. The hydrolytic products of oxidized AMP were identified as oxidized adenosine hemiacetals. Oxidized AMP was also found to be a non-competitive inhibitor with respect to p-nitrophenyl phosphate, with identical Kis and Kii values of 0.15 mM. Our results indicate that oxidized AMP could combine with the enzyme to form a binary complex, followed by reaction with the proximal lysyl amino group to yield a Schiff base. The latter was reduced with NaBH4 and identified by t.l.c. The incorporation of only 1.5 molecules of oxidized [14C]AMP per enzyme subunit resulted in a complete inactivation of the enzyme. The modified enzyme showed higher apparent Km for the substrates and higher Ki for inorganic phosphate, but lower [32P]phosphate incorporation, than the native enzyme. These results support the conclusion that a lysine residue is involved in the phosphate-binding site of human placental alkaline phosphatase.

Adenosine Monophosphate↗
PubMed1992

A Raman study of the binding of Fe(III) to ATP and AMP.

ATP-Fe and AMP-Fe complexes in water (H2O and 2H2O) at pH 7.5 were studied using Raman spectroscopy. Parallel and perpendicular polarization spectra were recorded in the spectral range 200-1650 cm-1, and the depolarization ratios for most of the bands were calculated. The changes in the frequencies, intensities and depolarization ratios of the ATP and AMP bands after the addition of FeCl3 showed that the adenine moiety, in addition to the phosphate(s), was involved in the binding of Fe to both ATP and AMP. Direct interactions of Fe(III) with the phosphate chain and the N-7 nitrogen and indirect interaction (via water molecules) with the amide group were proposed for the ATP-Fe complex. In contrast, direct interaction with the phosphate group and indirect interaction with the amide group were observed for the AMP-Fe complex. The different interactions of the two complexes suggest an 'anti' conformation for the ATP-Fe complex and a 'syn' conformation for the AMP-Fe complex. The strong binding of Fe to ATP compared with AMP and the difference in the conformation of the ATP-Fe and the AMP-Fe complexes may be significant in the pathway of Fe release in mitochondria.

Adenosine Monophosphate↗
PubMed1996

Cell shape change reveals the cyclic AMP-mediated action of follicle stimulating hormone, human chorionic gonadotrophin and vasoactive intestinal peptide in primary cultured human granulosa-lutein cells.

Granulosa cells are known to be the site of action of various hormones and agents that regulate ovarian function. This study was conducted to evaluate the effects of gonadotrophins, vasoactive intestinal peptide (VIP), prostaglandin (PG) F2 alpha and angiotensin II on the cyclic AMP (c-AMP) signalling transduction pathway in human granulosa-lutein cells. Exposure to agents that elevate c-AMP or mimic c-AMP action caused the cells to become rounded in a process that was rapid and reversible. We were able to demonstrate this cell rounding process in the presence of gonadotrophins and VIP, but not in the presence of PGF 2 alpha or angiotensin II. In addition, incubation of the cells with various selective phosphodiesterase (PDE) inhibitors revealed that the PDE type IV isoform, but not type III, catalyses c-AMP degradation in human granulosa-lutein cells. Alteration in c-AMP-dependent cytomorphology appears to be a convenient method to analyse the regulation of c-AMP-mediated events in the human granulosa-lutein cells.

Adenosine Monophosphate↗
PubMedDate not supplied

Timing and duration of administration are crucial for antiinfarct effect of AMP 579 infused at reperfusion in rabbit heart.

The adenosine A(1)/A(2) receptor agonist AMP 579 has been reported to protect the heart against infarction even when administered after the onset of ischemia. The present study explored both the timing and the duration of treatment required to limit infarct size in in situ rabbit hearts subjected to 30 min of regional ischemia and 3 hours of reperfusion. In groups 1 and 2, AMP 579 was infused from 10 min before reperfusion and continued for either 30 or 40 min. In group 3, AMP 579 was begun at the onset of reperfusion and continued for 70 min. In group 4, AMP 579 was also infused for 70 min but begun 10 min after reperfusion. In untreated hearts 36.4 +/- 3.1% of the risk zone infarcted. Protection was observed only in hearts having a 70-minute infusion of AMP 579 starting at reperfusion (13.0 +/- 1.9%, P < 0.05). Therefore, AMP 579 must be present at the moment of reperfusion and have a continued presence of more than 30 min thereafter to protect. Importantly, because AMP 579 can protect when administered up to the time of reperfusion, it likely prevents a reperfusion injury, and, therefore, has impressive clinical potential.

Animals↗
PubMed1976

Spin-labelled AMP - an activator of phosphorylase.

1. A spin-labelled AMP derivative and its diamagnetic analogue activate phosphorylase b in the same way, but do not activate phosphorylase a. 2. The electron-spin-resonance spectra of the spin-labelled AMP derivative bound to phosphorylase b and a have "powderlike" characteristics indicating that the spin label is immobilised on the protein. From changes in the electron-spin-resonance spectrum of spin-labelled AMP as phosphorylase b or a is added, the dissociation constants were calculated. 3. The interactions of spin-labelled AMP and the diamagnetic analogue with phosphorylase b and a have been monitored by observing changes in the spectral properties of fluorescent and spin-label probes covalently attached to the enzyme. 4. The dissociation constants of spin-labelled AMP and phosphorylase b or a are 175 +/- 25 muM and 15 +/- 5 muM respectively. Similar dissociation constants are obtained for the diamagnetic analogue. The effect of these AMP derivatives on the covalently attached probe groups and on phosphorylase activity is compared to the effect of AMP and IMP.

Adenosine Monophosphate↗
PubMed1976

AMP analogs: their function in the activation of glycogen phosphorylase b.

A series of AMP analogs has been selected in order to better understand the structural requirements (a) for the efficient binding of the activator molecule at the correct site on phosphorylase b from rabbit skeletal muscle and (b) for the activation which is observed. Two types of activation are known, according to Black and Wang [J. Biol. Chem. 243, 5892-5898 (1968)]: either a cooperative response with respect to the activator concentration (like the one which is obtained for AMP itself) or a non-cooperative response observed in the case of IMP. It is shown that the 5'-phosphate moiety is absolutely required for the analog to bind at the correct site (adenine or adenosine bind at another enzymic site), and that the free enthalpy, delta G, corresponding to the association process varies in a complex manner with respect to the substitution of the different positions of the AMP molecule. Moreover, the differences delta G (analog) - delta G (AMP) = delta G obtained for two types of substitution separately do not add up to the same energy difference as the one obtained when the two substitutions are made simultaneously on the AMP molecule. It appears that all the mononucleotides which have been tested up to now may be divided into two classes. Class I (AMP class) is characterized, apart from a strong activation, by the following features: (a) one molecule of analog expels two molecules of bound glucose 6-phosphate as it binds on the enzyme; (b) bound analog protects slowly one crucial cysteinyl residue against attack by 5,5'-dithio-bis(2-nitrobenzoic acid) at 4 degrees C; (c) association of two molecules of dimer is strengthened at 4 degrees C in the presence of the analog. Class II (IMP class) is associated with a weak activation and with the following set of properties: (a) a single molecule of bound glucose 6-phosphate is released as the first molecule of analog binds on the dimer; (b) two slowly reacting cysteinyl residues per subunit are immediately protected against 5,5'-dithio-bis(2-nitrobenzoic acid) by the binding of the analog at 4 degrees C; (c) the analog dissociates the low amount of tetramer which is present at 4 degrees C in the absence of AMP into two molecules of dimer. These results are discussed according to a plausible scheme of transconformations taking place in glycogen phosphorylase b, a model which has been derived earlier by relaxation studies.

Adenine↗
PubMed1992

Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets.

1. Acetyl-CoA carboxylase was purified to homogeneity, in the presence of protein phosphatase inhibitors, from rat liver sampled without freeze-clamping. The enzyme was in a highly phosphorylated state (4.8 mol/subunit) of low specific activity, and could be dramatically reactivated by treatment with protein phosphatase-2A. Amino acid sequencing and fast-atom-bombardment mass spectrometry showed that the enzyme was phosphorylated in Ser79, Ser1200 and Ser1215, the three sites known to be phosphorylated in cell-free assays by the AMP-activated protein kinase. 2. The inactive enzyme could also be completely reactivated using a limited treatment with trypsin, which removes the N-terminal segment containing Ser79 and reduces the phosphate content to 3.5 mol/subunit. These results strengthen previous findings that it is phosphorylation at Ser79 by the AMP-activated protein kinase that is responsible for the inactivation, and not the phosphorylation of the 220-kDa core fragment (which contains Ser1200 and Ser1215). 3. Analysis of the phosphorylation state of Ser79 in acetyl-CoA carboxylase from rat liver showed that phosphorylation occurs post mortem if freeze-clamping is not used. The higher phosphorylation observed in extracts made without freeze-clamping correlates with a large increase in AMP and decrease in ATP (presumably caused by hypoxia during removal of the liver), and with increased activity of the AMP-activated protein kinase. These results provide a rational explanation for the post mortem phosphorylation events, and re-emphasize the point that rapid cooling of cells and tissues is essential when measuring the expressed activity of acetyl-CoA carboxylase (as well as 3-hydroxy-3-methylglutaryl-CoA reductase). 4. Using the freeze-clamping procedure, the ratio of 'expressed' activity (measured in the presence of protein phosphatase inhibitors) to 'total' activity (measured after complete dephosphorylation) of rat liver acetyl-CoA carboxylase showed a marked diurnal rhythm, changing from 50% in the active form in the middle of the dark period to less than 10% active in the middle of the light period. The very low activity in the light period was associated with a high level of phosphorylation in Ser79. This diurnal rhythm is very similar to that previously described for the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase, another substrate for the AMP-activated protein kinase. Neither the activity of the AMP-activated protein kinase nor the content of AMP, ADP or ATP changed between the dark or light periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyl-CoA Carboxylase↗
PubMed1980

AMP metabolism in the marine bacterium Beneckea natriegens.

The catabolism of AMP by preparations from Beneckea natriegens has been reexamined. In the absence of ATP, cell-free extracts catabolized AMP via adenosine to inosine. When ATP was present, adenylate kinase converted AMP to ADP, lowering the rate of AMP catabolism. Particle-free supernatants (225,000 x g) metabolized AMP alone slowly, but adenylate kinase was active when ATP was added. Washed particulate fractions contained AMP nucleotidase activity which converted AMP to adenosine; in the presence of ATP, adenosine formation was reduced by residual adenylate kinase associated with the particulate fraction. IMP was not detected as a metabolite in these experiments.

5'-Nucleotidase↗