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PubMed1986

AMP nucleosidase: kinetic mechanism and thermodynamics.

The kinetic mechanism of AMP nucleosidase (EC 3.2.2.4; AMP + H2O----adenine + ribose 5-phosphate) from Azotobacter vinelandii is rapid-equilibrium random by initial rate studies of the forward and reverse reactions in the presence of MgATP, the allosteric activator. Inactivation-protection studies have established the binding of adenine to AMP nucleosidase in the absence of ribose 5-phosphate. Product inhibition by adenine suggests a dead-end complex of enzyme, AMP, and adenine. Methanol does not act as a nucleophile to replace H2O in the reaction, and products do not exchange into substrate during AMP hydrolysis. Thus, the reactive complex has the properties of concerted hydrolysis by an enzyme-directed water molecule rather than by formation of a covalent intermediate with ribose 5-phosphate. The Vmax in the forward reaction (AMP hydrolysis) is 300-fold greater than that in the reverse reaction. The Keq for AMP hydrolysis has been experimentally determined to be 170 M and is in reasonable agreement with Keq values of 77 and 36 M calculated from Haldane relationships. The equilibrium for enzyme-bound substrate and products strongly favors the enzyme-product ternary complex ([enzyme-adenine ribose 5-phosphate]/[enzyme-AMP] = 480). The temperature dependence of the kinetic constants gave Arrhenius plots with a distinct break between 20 and 25 degrees C. Above 25 degrees C, AMP binding demonstrates a strong entropic effect consistent with increased order in the Michaelis complex. Below 20 degrees C, binding is tighter and the entropic component is lost, indicating distinct enzyme conformations above and below 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1989

Structure and regulation of the AMP nucleosidase gene (amn) from Escherichia coli.

The gene for AMP nucleosidase from Escherichia coli (amn) has been sequenced and characterized. The gene codes for a transcript of 1.7 +/- 0.2 kb, and the open reading frame corresponds to a protein of 483 amino acids (Mr = 53848). Amino acid sequences from tryptic peptides of AMP nucleosidase, N-terminal amino acid analysis, and the amino acid composition confirm the gene assignment and the open reading frame of amn. Primer extension studies determined the 5'-end of the amn transcript. The 5'-regulatory region contains overlapping sequences with similarity to the consensus sequences for binding cAMP receptor protein and inorganic phosphate repressor protein. Addition of exogenous cAMP to E. coli deficient in adenylate cyclase resulted in a 3-fold increase in AMP nucleosidase activity. Growth of E. coli on limiting phosphate resulted in an 8-fold increase in the production of AMP nucleosidase. The amn gene was expressed in AMP nucleosidase deficient strains of Azotobacter vinelandii and E. coli. A pUC-amn construct is described that causes approximately 20% of the total protein in E. coli to be produced as AMP nucleosidase. Comparison of the amino acid sequence for AMP nucleosidase with that for yeast AMP deaminase indicates a region in which six of eight amino acids are identical but no other overall homology. The amino acid sequence showed poor agreement with consensus sequences for adenylate binding sites even though the enzyme is known to have a catalytic site for AMP and regulatory sites for MgATP and phosphate.

Amino Acid Sequence↗
PubMed1994

Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction.

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

Adenosine Monophosphate↗
PubMed2000

The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.

To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.

Adenosine Monophosphate↗
PubMed2001

Amp 579 reduces contracture and limits infarction in rabbit heart by activating adenosine A2 receptors.

To determine the mechanism by which AMP 579, an adenosine A1/A2 agonist, administered at reperfusion protects ischemic myocardium, buffer-perfused rabbit hearts were subjected to 30 min of global ischemia and 2 h of reperfusion. AMP 579 (500 nM) was included in the reperfusate for the first 70 min. Average left ventricular diastolic pressure during reperfusion in hearts receiving AMP 579 was lower than that in control hearts (17.9 +/- 2.4 vs. 39.0 +/- 6.5 mm Hg, p < 0.05), indicating attenuation of contracture. Left ventricular developed pressure and coronary flow during reperfusion were also significantly improved with AMP 579 treatment. AMP 579's anti-contracture effect was blocked by the adenosine A2-receptor antagonist 8-(3-chlorostyryl)caffeine (CSC), but not by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). CSC, but not DPCPX, also blocked AMP 579's ability to preserve developed pressure and coronary flow in these hearts. AMP 579 significantly reduced infarction in isolated hearts subjected to regional ischemia. The anti-infarct effect again was abolished by CSC but not by DPCPX. Finally, we tested whether 5'-(N-ethylcarboxamido)adenosine (NECA), another A1/A2 agonist, also administered for the initial 70 min of reperfusion, could duplicate the anti-infarct effect of AMP 579. One-hundred-nanomolar NECA duplicated the protection, but neither 50 nM CGS21680, a selective A2 agonist, nor 100 microM adenosine was protective. Therefore, AMP 579 given at reperfusion reduces contracture and infarction. Anti-contracture and anti-infarct effects require the adenosine A2, but not the A1, receptor suggesting that prevention of contracture and tissue salvage are mechanistically related. Not all A2 agonists were able to duplicate the anti-infarct effect, suggesting something unique about AMP579.

Animals↗
PubMed2002

Protection from AMP 579 can be added to that from either cariporide or ischemic preconditioning in ischemic rabbit heart.

AMP 579, an adenosine A /A receptor agonist, is cardioprotective when administered at reperfusion. Pretreatment with the Na /H exchanger inhibitor cariporide or ischemic preconditioning (PC) also limits infarct size. To gain insight into the mechanism of AMP 579 we investigated whether its protection could be added to that from either cariporide or PC. rabbit hearts were subjected to 45 min of regional ischemia followed by 3 h of reperfusion. Infarct size in the control group was 55.8 +/- 3.9% of the risk zone. PC significantly reduced infarct size to 26.0 +/- 6.7% (p<0.05). AMP 579 (30 micro g/kg) given just before reperfusion followed by 3 micro g/kg/min infusion for 70 min also limited infarct size (32.1 +/- 1.8%,) but the combination of AMP 579 and PC showed a significantly greater limitation of infarct size (5.5 +/- 2.7%, p < 0.05). Because cariporide pretreatment was so protective (8.5 +/- 3.7% infarction), we had to increase the ischemic insult to 60 min to test for any additive effect of the combination of AMP 579 + cariporide. Infarct size in the untreated group was 66.0 +/- 4.9% of the risk zone. Cariporide (0.5 mg/kg) 5 min prior to ischemia significantly reduced infarct size to 41.5 +/- 7.7%. When cariporide pre-treatment was combined with AMP 579 at reperfusion, infarction was further limited (14.2 +/- 4.5%). Because AMP 579's protection can be added to that of either cariporide or PC, AMP 579's mechanism of protection probably differs from either of them. The combination of AMP 579 + cariporide was particularly efficacious and could be useful in the surgical setting.

Animals↗
PubMed1993

Rapid turnover of the AMP-adenosine metabolic cycle in the guinea pig heart.

The intracellular flux rate through adenosine kinase (adenosine-->AMP) in the well-oxygenated heart was investigated, and the relation of the AMP-adenosine metabolic cycle (AMP<-->adenosine) to transmethylation (S-adenosylhomocysteine [SAH]-->adenosine) and coronary flow was determined. Adenosine kinase was blocked in isolated guinea pig hearts by infusion of iodotubercidin in the presence of the adenosine deaminase blocker erythro-9-(2-hydroxy-3-nonyl)adenine (5 mumol/L). Iodotubercidin (1 nmol/L to 4 mumol/L) caused graded increases in venous effluent concentrations of adenosine, from 8 +/- 3 to 145 +/- 32 nmol/L (mean +/- SEM, n = 3), and in coronary flow, which increased to maximal levels. Flow increases were completely abolished by adenosine deaminase (5 to 10 U/mL). Interstitial adenosine concentrations, estimated using a mathematical model, increased from 22 nmol/L during control conditions to 420 nmol/L during maximal vasodilation. The possibility that iodotubercidin caused increased venous adenosine by interfering with myocardial energy metabolism was ruled out in separate 31P nuclear magnetic resonance experiments. To estimate total normoxic myocardial production of adenosine (AMP-->adenosine<--SAH), the time course of coronary venous adenosine release was measured during maximal inhibition of adenosine kinase with 30 mumol/L iodotubercidin. Adenosine release increased more than 15-fold over baseline, reaching a new steady-state value of 3.4 +/- 0.3 nmol.min-1 x g-1 (n = 5) after 4 minutes. In parallel experiments, the relative roles of AMP hydrolysis and transmethylation (SAH hydrolysis) were determined, using adenosine dialdehyde (10 mumol/L) to block SAH hydrolase. In these experiments, adenosine release increased to similar levels of 3.4 +/- 0.5 nmol.min-1 x g-1 (n = 6) during inhibition of adenosine deaminase and adenosine kinase. It is concluded that (1) maximal increases in coronary flow are elicited by increases in interstitial adenosine concentration to approximately 400 nmol/L, (2) more than 90% of the adenosine produced in the heart is normally rephosphorylated to AMP without escaping into the venous effluent, (3) AMP hydrolysis is the dominant pathway for cardiac adenosine production under normoxic conditions, and (4) the high rate of adenosine salvage is due to rapid turnover of a metabolic cycle between AMP and adenosine. Rapid cycling may serve to amplify the relative importance of AMP hydrolysis over transmethylation in controlling cytosolic adenosine concentrations.

Adenosine↗
PubMed1975

The morphological transformation and inhibition of growth of bovine luteal cells in tissue culture induced by luteinizing hormone and dibutyryl cyclic AMP.

The luteal cells obtained from bovine corpus luteum by enzymatic treatment have been maintained in tissue culture. When the cells were maintained in the absence of luteinizing hormone or dibutyryl cyclic AMP, they grew parallel to one another and were elongated, thus giving to the culture a fibroblastic appearance. No contact inhibition was observed and the progestin secretion rate was low (3 pg per cell per day). In contrast, when luteinizing hormone or dibutyryl cyclic AMP was present, the cells became polygonal, growing as a monolayer and taking the appearance of epithelial cells. In this case contact inhibition was observed. The rate of progestin secretion was 250 pg per cell per day. As soon as luteinizing hormone or dibutyryl cyclic AMP was removed from the media, the cells reverted to a fibroblastic appearance. Agents such as colcemid, vinblastin or cytochalasin B inhibited the morphological effect of luteinizing hormone or dibutyryl cyclic AMP. Since those agents are known to inhibit the assembly of microtubules, the data suggest that LH and dibutyryl cyclic AMP act by promoting the organization of microtubules from protein monomers. This microtubular system (cytoskeleton) is responsible for the morphological appearance of the cells. Concomitant with the morphological changes induced by luteinizing hormone and dibutyryl cyclic AMP an inhibition in the growth rate of luteal cells was observed. It suggests that by raising the intracellular level of cyclic AMP the luteinizing hormone inhibits the division of luteal cells and is not, for that reason, a mitogenic agent. A similar effect was obtained with other agents known to stimulate cyclic AMP production such asthe prostaglandins. Steroids such as glucocorticoids and testosterone but not progesterone also inhibited the growth rate. It is concluded that luteinizing hormone by controlling the level of cyclic AMP within the luteal cells is responsible for the expression of the phenotype of the cells and the maintenance of differentiation.

Animals↗
PubMed2005

Antimicrobial peptides (AMPs): peptide structure and mode of action.

Antimicrobial peptides (AMPs) have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum. Their amino acid composition, amphipathicity, cationic charge, and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Although these models are helpful for defining mechanisms of AMP activity, their relevance to resolving how peptides damage and kill microorganisms still needs to be clarified. Moreover, many AMPs employ sophisticated and dynamic mechanisms of action to carry out their likely roles in antimicrobial host defense. Recently, it has been speculated that transmembrane pore formation is not the only mechanism of microbial killing by AMPs. In fact, several observations suggest that translocated AMPs can alter cytoplasmic membrane septum formation, reduce cell-wall, nucleic acid, and protein synthesis, and inhibit enzymatic activity. In this review, we present the structures of several AMPs as well as models of how AMPs induce pore formation. AMPs have received special attention as a possible alternative way to combat antibiotic-resistant bacterial strains. It may be possible to design synthetic AMPs with enhanced activity for microbial cells, especially those with antibiotic resistance, as well as synergistic effects with conventional antibiotic agents that lack cytotoxic or hemolytic activity.

Animals↗
PubMed2000

AMP decreases the efficiency of skeletal-muscle mitochondria.

Mitochondrial proton leak in rat muscle is responsible for approx. 15% of the standard metabolic rate, so its modulation could be important in regulating metabolic efficiency. We report in the present paper that physiological concentrations of AMP (K(0.5)=80 microM) increase the resting respiration rate and double the proton conductance of rat skeletal-muscle mitochondria. This effect is specific for AMP. AMP also doubles proton conductance in skeletal-muscle mitochondria from an ectotherm (the frog Rana temporaria), suggesting that AMP activation is not primarily for thermogenesis. AMP activation in rat muscle mitochondria is unchanged when uncoupling protein-3 is doubled by starvation, indicating that this protein is not involved in the AMP effect. AMP activation is, however, abolished by inhibitors and substrates of the adenine nucleotide translocase (ANT), suggesting that this carrier (possibly the ANT1 isoform) mediates AMP activation. AMP activation of ANT could be important for physiological regulation of metabolic rate.

Adenosine Monophosphate↗
PubMed2002

[In vitro evaluation on the effects of combined lamivudine (3TC) and Ara-AMP against hepatitis B virus].

BACKGROUND: To evaluate the inhibitive effect of combination of 3TC with Ara-AMP against HBV in vitro. METHODS: Used 2.2.15 cell as target cell. With radioimmunological technique and blot slot, the inhibitive effect of 3TC, Ara-AMP and the combination of both against the HBsAg, HBeAg and intracellular HBV DNA were investigated. RESULTS: The inhibitive ratio of Ara-AMP against HBsAg, HBeAg was 45.48% and 41.46% respectively when its concentration was 400.0 microgram/ml. Although 3TC also has inhibitive effect in its experimental concentration, its effect is weaker. When Ara-AMP 50.0 microgram/ml was combined with 3TC 1.25 and 5.00 microgram/ml respectively, the inhibitive ratio against HBsAg were 19.92% and 17.32% respectively. Compared with using same concentration 3TC alone, the difference of results was significant (P<0.05). But when compared with using the same concentration Ara-AMP alone, the difference of results had no statistical significance (P <0.05). Remarkable inhibitive effects of combination of 3TC with Ara-AMP against HBeAg were n ot found. When 3TC 5.00 microgram/ml was combined with Ara-AMP 12.5 and 50.0 microgram/ml respectively, the inhibitive ratio against HBV DNA was 45.90% and 50.36% respectively. Comparing the content of HBV DNA in these groups with that of control group and the groups using the same concentration 3TC and Ara-AMP alone, the differences were significant (P <0.05). CONCLUSIONS: Combination of 3TC with Ara-AMP could enhance the inhibitive effects against HBV DNA.

Antiviral Agents↗
PubMed1992

Analysis of the formation of AMP-DNA intermediate and the successive reaction by human DNA ligases I and II.

DNA ligation catalyzed by all DNA ligases involves two intermediary steps, the formation of the ligase-AMP and the AMP-DNA complexes. A method was developed to purify and analyze the AMP-DNA intermediate from the DNA ligation reaction catalyzed by DNA ligases. This AMP-DNA complex was maximally accumulated by preincubation of human DNA ligase I or II with ATP, followed by interaction with the DNA substrate for 5 s at 0 degrees C. The gel-purified AMP-DNA complex maintained its property as a ligation intermediate. The AMP was directly linked to the 5'-phosphate of DNA with a pyrophosphate bond. The successive ligation reaction following the AMP-DNA complex formation required DNA ligase and Mg2+ ion but was inhibited by ATP and pyridoxal 5'-phosphate, indicating that the availability of the AMP binding site in the enzyme is essential for the completion of the reaction. Furthermore, the formation of the AMP-DNA complex and the subsequent DNA ligation were substrate specific for human DNA ligases I and II. These data, together with previously reported results, suggest that a major difference between human DNA ligases I and II is in their DNA-binding domains. The methods make it convenient to study in depth the kinetics of the overall DNA ligation.

Adenosine Monophosphate↗
PubMed1983

The interaction of fructose 2,6-bisphosphate and AMP with rat hepatic fructose 1,6-bisphosphatase.

The binding of the inhibitory ligands fructose 2,6-bisphosphate and AMP to rat liver fructose 1,6-bisphosphatase has been investigated. 4 mol of fructose-2,6-P2 and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-bisphosphate exhibits negative cooperatively as indicated by K'1 greater than K'2 greater than K'3 greater than or equal to K'4 and a Hill plot, the curvature of which indicates K'2/K'1 less than 1, K'3/K'2 less than 1, and K'4/K'3 = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K'1 less than K'2 less than K'3 less than K'4 and an nH of 2.05. Fructose 2,6- and fructose 1,6-bisphosphates enhance the binding of AMP as indicated by an increase in the intrinsic association constants. At pH 9.2, where fructose 2,6-bisphosphate and AMP inhibition of the enzyme are diminished, fructose 2,6-bisphosphate binds with a lower affinity but in a positively cooperative manner, whereas AMP exhibits half-sites reactivity with only 2 mol of AMP bound per mol of tetramer. Ultraviolet difference spectroscopy confirmed the results of these binding studies. The site at which fructose 2,6-bisphosphate binds to fructose 1,6-bisphosphatase has been identified as the catalytic site on the basis of the following. 1) Fructose 2,6-bisphosphate binds with a stoichiometry of 1 mol/mol of monomer; 2) covalent modification of the active site with acetylimidazole inhibits fructose 2,6-bisphosphate binding; and 3) alpha-methyl D-fructofuranoside-1,6-P2 and beta-methyl D-fructofuranoside-1,6-P2, substrate analogs, block fructose 2,6-bisphosphate binding. We propose that fructose 2,6-bisphosphate enhances AMP affinity by binding to the active site of the enzyme and bringing about a conformational change which may be similar to that induced by AMP interaction at the allosteric site.

Adenosine Monophosphate↗
PubMed1998

Cardioprotective effects of the novel adenosine A1/A2 receptor agonist AMP 579 in a porcine model of myocardial infarction.

This study examined the cardioprotective effects and pharmacology of the novel adenosine A1/A2 receptor agonist ([1S-[1a,2b,3b, 4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]amino]-3H-imida zo[4,5-b] pyridyl-3-yl] cyclopentane carboxamide) (AMP 579), in a model of myocardial infarction. Experiments were performed in pentobarbital-anesthetized pigs in which myocardial infarction was induced by a 40-min occlusion of the left anterior descending coronary artery, followed by 3 hr of reperfusion. This procedure resulted in approximately 20% of the left ventricle being made ischemic in all test groups. In untreated animals, an infarct size equal to 56 +/- 5% of the ischemic area was observed. Preconditioning, with two cycles of 5 min of ischemia followed by 10-min reperfusion, resulted in a 70% reduction in infarct size (17 +/- 5%) relative to risk area. Administration of AMP 579 30 min before ischemia (3 microg/kg i.v. followed by 0.3 microg/kg/min i.v. through 1 hr of reperfusion) did not change blood pressure, HR or coronary blood flow but resulted in marked cardioprotection: a 98% reduction in infarct size (1 +/- 1%) relative to risk area. Moreover, whereas approximately 90% of control pigs suffered ventricular fibrillation during ischemia, no fibrillation was observed in animals treated with AMP 579. Further experiments determined the effects of AMP 579 when administered 30 min after the onset of myocardial ischemia, 10 min before reperfusion. Two doses were studied: a low hemodynamically silent dose (3 microg/kg + 0.3 microg/kg/min through 1 hr of reperfusion) and a 10-fold higher dose that did cause reductions in blood pressure and HR. Both doses of AMP 579 produced a comparable cardioprotective effect, reducing infarct size to approximately 50% of that observed in control animals. The cardioprotective effect of AMP 579 was a consequence of adenosine receptor stimulation, because it was completely inhibited by pretreatment with the specific adenosine receptor antagonist CGS 15943 (1 mg/kg i.v.). However, the selective A1 receptor agonist GR 79236 (3 microg/kg + 0.3 microg/kg/min i.v.) did not reduce infarct size, which suggests that under these experimental conditions, stimulation of adenosine A2 receptors is important for the cardioprotective effect of AMP 579. The adenosine-regulating agent acadesine (5 mg/kg + 0.5 mg/kg/min i.v.) also failed to reduce infarct size. In conclusion, the novel adenosine A1/A2 receptor agonist AMP 579 produces marked cardioprotection whether administered before myocardial ischemia or reperfusion. Cardioprotection is not dependent on changes in afterload or myocardial oxygen demand and is a consequence of adenosine receptor stimulation. The pharmacological profile of AMP 579 in this model is consistent with its potential utility in the treatment of acute myocardial infarction.

Acute Disease↗
PubMed1987

Ecto-cyclic AMP-receptor in goat epididymal intact spermatozoa and its change in activity during forward motility.

Goat epididymal intact spermatozoa have been shown to possess on the external surface specific receptors that bind with high affinity to exogenous [8-3H]cyclic AMP. The ecto-cyclic AMP-receptor activity was not due to contamination of broken or "leaky" cells, if any. The binding reaction of [3H]cyclic AMP with the receptors was extremely rapid. Uptake of the labeled cyclic AMP to the sperm cytosolic fraction was undetectable. There was little leakage of cyclic AMP-receptors from intact spermatozoa during the binding assays. The binding reaction was proportional to cell concentration, specific and saturable at 250 nM cyclic AMP. The binding of the labelled cyclic nucleotide was nearly completely displaced at saturating concentrations (2.5 microM) of the unlabelled nucleotide. The ecto-receptors showed high specificity for binding to cyclic AMP. The Kd of the binding sites was approximately 1.7 X 10(-8) M. The binding interaction was highly sensitive to treatment with proteolytic enzymes: trypsin, chymotrypsin, or pronase (125 micrograms/ml). Sonication caused a nearly 450% increase of the ecto-receptor activity. The specific activity of the ecto-cyclic AMP-receptor was approximately twofold higher in the vigorously forwardly motile spermatozoa than in the "composite" cells, suggesting that the ecto-receptors may have a role in modulating flagellar motility.

Animals↗
PubMed1975

Somatic genetic analysis of cyclic AMP action: characterization of unresponsive mutants.

N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate kills cultured mouse lymphosarcoma cells, but not resistant mutants derived by a single-step clonal selection. Resistant clones lack the cyclic AMP binding proteins present in wild type, cyclic AMP sensitive clones. Both endogenous cyclic AMP, accumulated in response to isoproterenol or cholera toxin, and exogenous dibutyryl cyclic AMP induce cyclic AMP phosphodiesterase, slow growth, and eventually kill wild type cells. In the resistant mutants, however, the endogenous and exogenous cyclic nucleotides appear to be completely inactive. These results indicate that an intracellular receptor for cyclic AMP, previously shown to be associated with a cyclic AMP-dependent protein kinase, mediates cyclic AMP's regulation of growth and phosphodiesterase synthesis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Alpha 2-adrenoceptor mediated inhibition of forskolin-stimulated cyclic AMP accumulation in isolated porcine palmar lateral veins.

The aim of this study was to use a 3H-adenine pre-labelling technique to characterise the effect of alpha 2-adrenoceptor activation on forskolin-stimulated cyclic AMP accumulation in the isolated porcine palmar lateral vein. Forskolin (10(-7)-10(-4) M) stimulated 3H-cyclic AMP accumulation in the isolated porcine palmar lateral vein in a biphasic and concentration-dependent manner. In the absence of the cyclic AMP-selective phosphodiesterase inhibitor rolipram, forskolin stimulated 3H-cyclic AMP accumulation approximately 7-8 fold. The response reached a peak after 5 min. In the presence of rolipram (10(-5) M), basal 3H-cyclic AMP levels were approximately 70% higher than in its absence (basal: 1823 +/- 57 dpm; rolipram: 3088 +/- 229, n = 3) and forskolin (3 x 10(-5) M) stimulated 3H-cyclic AMP accumulation approximately 8 fold. The latter response reached a plateau 10 min after the addition of forskolin. In all subsequent studies, the tissues were incubated with forskolin (3 x 10(-5) M) for 5 min in the absence of rolipram. Noradrenaline (NA; 10(-9)-10(-4) M) and UK14304 (10(-9)-10(-4) M) inhibited forskolin-stimulated 3H-cyclic AMP accumulation in a concentration-dependent manner with mean pIC50 values of 7.61 +/- 0.37 (n = 4) and 7.76 +/- 0.23 (n = 5), respectively. With either NA or UK14304, the maximal inhibition of the forskolin response obtained was approximately 75%. Neither NA (10(-4) M) nor UK14304 (10(-4) M) altered basal 3H-cyclic AMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1978

Receptor-linked cyclic AMP systems in rat neostriatum: differential localization revealed by kainic acid injection.

Various receptor-linked cyclic AMP systems were measured in rat neostriatum 2--14 days after selective destruction of neuronal cell bodies and dendrites by micro-injection of 3 microgram of kainic acid. Basal adenylate cyclase activity was reduced by up to 56% in the injected side and the sensitivity to dopamine was abolished. Up to 84% of cyclic nucleotide phosphodiesterase activity, hydrolyzing either cyclic AMP or cyclic GMP, was destroyed by kainic acid injection. Specific binding of [3H]etorphine and [3H]spiroperidol was reduced by up to 62% in the injected side, while non-specific binding was unchanged. All of these changes were time-dependent, and were greatest 7--14 days after kainic acid treatment. On the other hand, intrastriatal kainic acid injection caused no change in the steady-state concentration of cyclic AMP in striatal slices, or in the in vivo cyclic AMP content in the striatum of rats killed by microwave irradiation. Receptor-mediated increases in cyclic AMP accumulation in striatal slices were either unchanged or markedly potentiated by kainic acid treatment. The maximum response to adenosine was unchanged, while the response to isoprenaline was increased up to 3.7-fold, the response to dopamine increased up to 6.7-fold, and the response to PGE1 increased up to 30-fold. The effect of dopamine in kainic acid-treated striatal slices was no longer blocked by fluphenazine, but was blocked by propranolol, suggesting an interaction of dopamine with a beta-adrenoceptor in kainic acid-treated slices. The results suggest differential cellular localizations of the various receptor-linked cyclic AMP systems in rat neostriatum. Some dopamine and opiate receptors, as well as most of the phosphodiesterase activity, are associated with local neuronal elements, while beta-adrenoceptor, adenosine and PGE1 alterations in cyclic AMP are not. The potentiation of the beta-adrenoceptor and PGE1 responses suggests that they may occur in glial cells. In addition, the pool of adenylate cyclase destroyed by kainic acid appears to make little contribution to normal levels of cyclic AMP in the tissue.

3',5'-Cyclic-AMP Phosphodiesterases↗