Normal Adenine Research Articles

Demethylation-Driven ligase chain reaction for simultaneously sensitive detection of m6A demethylase FTO and m1A demethylase ALKBH3 in breast cancer at Single-Molecule level

RNA demethylases play a crucial role in gene expression regulation by controlling RNA methylation levels, and their abnormal expressions are associated with many human diseases. Herein, we develop a single-molecule detection method for simultaneous quantification of N6-methyladenosine (m6A) demethylase FTO and N1-methyladenosine (m1A) demethylase ALKBH3 based on RNA demethylation-directed multi-cyclic ligase chain reaction (LCR) cascades. We design a ssDNA containing m6A modification as the substrate of FTO and a ssDNA containing m1A modification as the substrate of ALKBH3. FTO and ALKBH3 catalyze the demethylation of m6A and m1A to generate normal adenine (A), respectively. Afterwards, Taq ligase initiates the LCR reaction with the demethylated ssDNAs as the templates to produce abundant biotinylated Cy3/Cy5-labeled signal probes. Subsequently, the resultant biotinylated probes are captured by streptavidin-coated magnetic beads (MBs) to form the MB-ssDNA-Cy3/Cy5 complexes. After magnetic separation, the ssDNAs in the MB-ssDNA-Cy3/Cy5 complexes are digested by DNase I, liberating abundant Cy3 and Cy5 fluorophores that can be counted by single-molecule detection. Remarkably, only a single DNA ligase is employed to achieve target signal conversion and multi-cyclic cascade amplification. This method displays extremely high sensitivity with a limit of detection of 6.33 × 10−18 mol/L for FTO and 1.44 × 10−17 mol/L for ALKBH3. Moreover, it can measure the enzyme kinetic parameters, screen inhibitors, and quantify cellular FTO and ALKBH3 activities with single-cell sensitivity. Furthermore, it can differentiate the FTO and ALKBH3 levels in the tissues of healthy human and breast cancer patients, offering a robust platform for biomedical research and clinical diagnosis.

DNA Deformation Exerted by Regulatory DNA-Binding Motifs in Human Alkyladenine DNA Glycosylase Promotes Base Flipping.

Human alkyladenine DNA glycosylase (AAG) is a key enzyme that corrects a broad range of alkylated and deaminated nucleobases to maintain genomic integrity. When encountering the lesions, AAG adopts a base-flipping strategy to extrude the target base from the DNA duplex to its active site, thereby cleaving the glycosidic bond. Despite its functional importance, the detailed mechanism of such base extrusion and how AAG distinguishes the lesions from an excess of normal bases both remain elusive. Here, through the Markov state model constructed on extensive all-atom molecular dynamics simulations, we find that the alkylated nucleobase (N3-methyladenine, 3MeA) everts through the DNA major groove. Two key AAG motifs, the intercalation and E131-N146 motifs, play active roles in bending/pressing the DNA backbone and widening the DNA minor groove during 3MeA eversion. In particular, the intercalated residue Y162 is involved in buckling the target site at the early stage of 3MeA eversion. Our traveling-salesman based automated path searching algorithm further revealed that a non-target normal adenine tends to be trapped in an exo site near the active site, which however barely exists for a target base 3MeA. Collectively, these results suggest that the Markov state model combined with traveling-salesman based automated path searching acts as a promising approach for studying complex conformational changes of biomolecules and dissecting the elaborate mechanism of target recognition by this unique enzyme.

Characterization of substrate binding and enzymatic removal of a 3-methyladenine lesion from genomic DNA with TAG of MDR A. baumannii.

The rise of multiple-drug resistance in bacterial pathogens imposes a serious public health concern and has led to increased interest in studying various pathways as well as enzymes. Different DNA glycosylases collaborate during bacterial infection and disease by overcoming the effects of ROS- and RNS-mediated host innate immunity response. 3-Methyladenine DNA glycosylase I, an essential DNA repair enzyme, was chosen for the present study from the MDR species of A. baumannii. The enzyme was especially chosen because of its functional significance in A. baumannii and due to its structural variation from its human homologue. MDR strains such as A. baumannii are interesting targets owing to their evolved mechanisms of evading a host defence. In the absence of any structural information, the enzyme was characterized biophysically and biochemically. Binding studies with 3mA and Zn2+ indicated that the activity of TAG-Ab is an enthalpy-driven process. Fluorescence thermal denaturation studies described that the denaturation of TAG-Ab is a two-step process. Modified RP-HPLC-based glycosylase assay attested that the heterologously expressed and purified TAG-Ab enzyme is active and catalyses the removal of 3mA. Other binding parameters and the effect of adenine on substrate binding are also discussed in detail.

Structural Basis of DNA Loop Recognition by Endonuclease V

The DNA repair enzyme endonuclease V (EndoV) recognizes and cleaves DNA at deaminated adenine lesions (hypoxanthine). In addition, EndoV cleaves DNA containing various helical distortions such asloops, hairpins, and flaps. To understand the molecular basis of EndoV's ability to recognize and incise DNA structures with helical distortions, we solved the crystal structure of Thermotoga maritima EndoV in complex with DNA containing a one-nucleotide loop. The structure shows that a strand-separating wedge is crucial for DNA loop recognition, with DNA strands separated precisely at the helical distortion. The additional nucleotide forming the loop rests on the surface of the wedge, while the normal adenine opposite the loop is flipped intoa base recognition pocket. Our data show a different principle for DNA loop recognition and cleavage byEndoV, in which a coordinated action of a DNA-intercalating wedge and a base pocket accommodating a flipped normal base facilitate strand incision.

Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA

To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (AT) and its photophysical characterization inside DNA. AT shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, AT shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that AT only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that AT shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, AT shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.

Evaluation of Platelets Obtained from Premature Neonates To Determine Whether Platelet Dysfunction Mediates the Development of Intraventricular Hemorrhage.

Hemorrhagic complications occur in neonates, including intraventricular hemorrhage (IVH) in premature babies. The premature infant appears to be at greatest risk for a head bleed from days 2–3 until day 7 after birth. A relationship exists that suggests that the shorter the gestation, the greater the risk for this potentially fatal hemorrhagic event. The basis of intra-ventricular brain hemorrhage (IVH) in the premature infant is unknown. Premature birth is not necessarily the cause of IVH. The neonate is known to have a transient platelet dysfunction at the time of birth. Studies to define the transient platelet dysfunction in the neonate have been limited. Explanations in the literature include:a diminished response to physiologic agonists,defective platelet (PL) dense granule (DG) secretion, and/orineffective mobilization of intracellular calcium in platelets.We and others have found that the adenine nucleotide content of platelets obtained from umbilical cord blood is significantly less than seen in adults. We have found that platelets obtained from umbilical cord blood have an average of 0.79±0.08 DG/PL (n=62), which is significantly decreased compared to the adult population (4–6 DG/PL) (Blood , 100(11):691a, 2002). Although the concentration of ADP and ATP were found to be decreased compared to normal adult platelets, the ATP/ADP ratio in neonatal platelets is 1.95 (n=61), consistent with established adult normal range ratio values (1.4–1.95). The current studies are ultimately intended to evaluate infants that have survived severe IVH for platelet dysfunction due to platelet dense granule deficiency (δ-SPD). Preliminary data have been obtained on peripheral blood from premature infants (n=15) obtained 3–5 d after birth. Their values (mean±2sd) are: gestational age 31±4.8 weeks and wt 1506±474 gr. The DG number (1.01±0.16/PL) is significantly decreased compared to normal adults, while the ATP (3.19±0.36 μM/1011 PL), ADP (2.63±0.54 μM/1011 PL) and ATP/ADP ratio (1.46±0.13) are normal. The decreased DG number in premature PL is strikingly similar to the DG content in cord blood PLs. We have studied one infant's cord blood (2.41 DG/PL)and day 1 peripheral blood (3.87 DG/PL). The parents have had a normal DG number and adenine nucleotide content. These results would suggest that the premature infant platelets may have an incomplete incorporation of calcium into the dense granules 3–5 days after birth when compared to the normal adenine nucleotide content. A decrease in DG calcium would explain the decreased numbers of DGs calculated from whole mounted PLs by EM. Calcium is electron dense and if lacking in DGs, would result in an artificially low number of DGs determined by this method. To date, we have not obtained blood from a premature infant with a significant IVH or its parents to evaluate δ-SPD as a potential etiology of severe IVH.

Decreased Dense Granule Volume in Neonatal Platelets.

Two models of biogenesis have been proposed for the development of platelet granules. One model postulates that granule contents develop in the maturing megakaryocyte via a direct biosynthetic pathway from the golgi, whereas the second model is thought to involve the multivesicular body for sorting and targeting granular constituents via an endosomal pathway. While these models may appropriately apply to the mechanism of the development of platelet alpha granules, dense granule biosynthesis is not readily explained. In animal studies and in human cultured megakaryocyte experiments, molecular markers (CD63 and serotonin) have been reported to appear concomitant with alpha granule formation, yet the characteristic density of the delta granule is not present until later in the maturation of the megakaryocyte. This suggests that dense granule vesicles must be “loaded” for a fully functioning platelet. Our evaluations of platelets obtained from umbilical cord blood would suggest that circulating platelets may become fully functional after birth via a mechanism that loads specific vesicles. The neonate is known to have a transient platelet dysfunction at the time of birth which has been explained in the literature as being due to 1) a diminished response to physiologic agonists, 2) defective platelet dense granule secretion, and/or 3) ineffective mobilization of intracellular calcium in platelets. Studies to define the transient platelet dysfunction in the neonate have been limited. We and others have found that the adenine content of platelets obtained from umbilical cord blood is significantly less than seen in adults. We have found that platelets obtained from umbilical cord blood have an average of 0.79 ± 0.08 DG/PL (n=62), which is significantly decreased compared to the adult population and that the ATP:ADP ratio in these platelets is 1.95 (n=61), consistent with established adult normal range ratio values (1.4–1.95). The actual measured adenine content was found to be decreased in the neonatal platelets (ATP = 2.76 ± 0.53 mmol/1011 PL; ADP=0.96±0.19 mmol/1011 PL). The normal adenine nucleotide DG contents have been reported to be 3.5–7 mmol/1011 PL for ATP and 1.9–3.7 mmol/1011 PL for ADP in adults. Using the uranaffin reaction technique to classify the four types (I–IV) of dense granules present in neonatal platelets, we have found a significant difference in the percentage of each type than is found in adult controls. We have employed an image analysis technique (Blood , 90: 140a, 1997) to calculate the mean volume of dense granules in neonatal platelets. We report that not only do neonatal platelets have a deficiency of dense granules, but that these granules are also extremely small in size having an average volume of 6.45 X 105 nm3 (n =36) (normal adult = 8–12 X 106 nm3). These results would suggest that the neonatal platelet has an incomplete incorporation of constituents (i.e., calcium, ADP) into the dense granules at the time of birth and that an unknown mechanism exists that induces dense vesicle “loading” at some time after birth. These observations may prove useful in pursuit of understanding the mechanism of platelet dense granule biosynthesis.

Neonatal hepatic steatosis by disruption of the adenosine kinase gene.

Neonatal hepatic steatosis (OMIM 228100) is a fatal condition of unknown etiology characterized by a pale and yellow liver and early postnatal mortality. In the present study, a deficit in adenosine-dependent metabolism is proposed as a causative factor. Physiologically, adenosine is efficiently metabolized to AMP by adenosine kinase (ADK), an enzyme highly expressed in liver. ADK not only ensures normal adenine nucleotide levels but also is essential for maintaining S-adenosylmethionine-dependent transmethylation processes, where adenosine, an obligatory product, has to be constantly removed. Homozygous Adk(-/-) mutants developed normally during embryogenesis. However, within 4 days after birth they displayed microvesicular hepatic steatosis and died within 14 days with fatty liver. Adenine nucleotides were decreased and S-adenosylhomocysteine, a potent inhibitor of transmethylation reactions, was increased in the mutant liver. Thus, a deficiency in adenosine metabolism is identified as a powerful contributor to the development of neonatal hepatic steatosis, providing a model for the rapid development of postnatally lethal fatty liver.

Functional Interaction of MutY Homolog with Proliferating Cell Nuclear Antigen in Fission Yeast, Schizosaccharomyces pombe

The MutY homolog (MYH) is responsible for removing adenines misincorporated on a template DNA strand containing G or 7,8-dihydro-8-oxoguanine (8-oxoG) and thus preventing G:C to T:A mutations. Human MYH has been shown to interact physically with human proliferating cell nuclear antigen (hPCNA). Here, we report that a similar interaction between SpMYH and SpPCNA occurs in the fission yeast Schizosaccharomyces pombe. Binding of SpMYH to SpPCNA was not observed when phenylalanine 444 in the PCNA binding motif of SpMYH was replaced with alanine. The F444A mutant of SpMYH expressed in yeast cells had normal adenine glycosylase and DNA binding activities. However, expression of this mutant form of SpMYH in a SpMYHDelta cell could not reduce the mutation frequency of the cell to the normal level. Moreover, SpMYH interacted with hPCNA, and SpPCNA interacted with hMYH but not with F518A/F519A mutant hMYH containing mutations in its PCNA binding motif. Although the SpMYHDelta cells expressing hMYH had partially reduced mutation frequency, the F518A/F519A mutant hMYH could not reduce the mutation frequency of SpMYHDelta cells. Thus, the interaction between SpMYH and SpPCNA is important for SpMYH biological function in mutation avoidance.

Determination of O6-Methylguanine in DNA by High-Performance Liquid Chromatography with Electrochemical Detection.

O6-Methylguanine (O6-MeGua), formed in calf thymus DNA by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitrosourea (MNU) in vitro was determined by high-performance liquid chromatography (HPLC) with electrochemical detection. The DNA treated with MNNG or MNU was hydrolyzed to nucleic bases under a mild acidic condition. O6-MeGua was separated from abundant normal adenine and guanine bases with a Sep-Pak C18 cartridge prior to HPLC analysis. With the cartridge, the peak areas of adenine and guanine bases decreased less than 0.3% as compared to those without clean-up process, although the recovery of O6-MeGua through the clean-up process was more than 96%. The amount of O6-MeGua formed from 1μg of DNA treated with 7.3 nmol of MNNG and 14.7 nmol of MNU was 9.66 and 11.1 pmol, respectively.

Adenine nucleotide metabolism and contractile dysfunction in heart failure--biochemical aspects, animal experiments, and human studies.

In myocardial hypertrophy and heart failure a series of adaptational changes occur some multiplying contractile units, others slowing shortening velocity and increasing economy of contraction. The demonstration of energy-saving mechanisms in heart failure has prompted further investigations of energy providing and utilizing metabolic pathways. The use of myocardial ATP as a substrate occurs mainly at the myosin-ATPase and at the Ca-ATPase of the sarcoplasmic reticulum. As the Michaelis constant of both enzymes for ATP is in the micromolar (microM) range, whereas cellular ATP content is about 5000 microM, these enzymes are not controlled by the availability of ATP as a substrate. In experimental heart failure in large animals, normal or reduced creatine phosphate levels (in most cases together with normal adenine nucleotides) have been described. Reduced creatine phosphate is found in models with increased oxygen consumption, and creatine phosphate may buffer the ATP pool in these models. In human heart failure due to dilated cardiomyopathy, where resting oxygen consumption per unit mass and lactate extraction are normal in most patients, normal adenine nucleotides, creatine phosphate, and mitochondrial function have been described in the initial studies. These results have been challenged by one study showing decreased ATP levels in dilated cardiomyopathy, correlating with the decrease in ejection fraction. However, only ATP has been measured in this study, whereas total adenine nucleotides may be a more suitable parameter. Recently published results have again demonstrated normal ATP and total adenine nucleotides in human heart failure. In the same patients, significantly decreased myocardial norepinephrine was measured, indicating that metabolic changes had occurred in these hearts, but were independent of adenine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

The biochemical basis of sickle cell anemia in Saudi Arabia: A model case of genetic and molecular disorders

2 THE BIOCHEMICAL BASIS OF SICKLE CELL ANEMIA IN SAUDI ARABIA: A MODEL CASE OF GENETIC AND MOLECULAR DISORDERS Introduction Fetal Hemoglobin A I ALAYASH AND J BONAVENTURA Duke University Marine Biomedical Center Beaufort, North Carolina 28516-9721 USA Sickle cell anemia is a genetic disorder that appeared as an isolated mutation in equatorial Africa and spread to some parts of the Arabian peninsula, to Southern Europe and to North America as a result of population movement. 1 Its molecular basis is well understood and can be described as follows: When oxygen tension is reduced, the deoxygenated sickle cell hemoglobin (HbS) molecules undergo polymerization that leads to the formation of long fibers which cause the red blood cell to assume a sickle-like shape. 2 This polymerization is attributed to the replacement of a thymine nucleotide for the normal adenine nucleotide in the sixth codon of the 13 chain which results in substitution of valine for glutamic acid. 3 This amino acid residue lies on the outside of the molecule and does not participate in oxygen binding. Oxygenation and deoxygenation cause little tertiary change in the part of the molecule where this residue is located. In the deoxy form, however, the overall shape of the HbS molecule is such that the mutant valine is capable of forming new hydrophobic bonds with leucine and phenylalanine residues in positions 85 and 88 of an adjacent 13 chain of another HbS molecule. 4 Only the valine residue of one of the two 13 chains in each HbS molecule in the polymer is involved in such linkage. HbS now has a changed electrophoretic mobility and reduced solubility upon oxygenation. 5 Homozygotes for the HbS gene suffer from sickle cell disease and frequently have episodes of severe pain, part of which is caused by the inability of the sickle-shaped cells to be flexible enough to pass through the narrow capillaries. This rigidity causes the cells to block the capillaries thus preventing normal blood flow or causes the cells to break at normal blood pressure. 6 Homozygotes for the HbS gene of African origin have a reduced life expectancy] In contrast, those of non-African origin are known to survive to an advanced age. One such group is the Saudi Shiites who inhabit the Alqatif and Alhassa oases of the Eastern Province of Saudi Arabia where the HbS gene occurs in 20-30% of the population. Saudi Arabs who have sickle cell anemia exhibit a mild course for this disease. 8 HbS isolated from them has the identical 13-chain replacement of glutamic acid by valine at position six. 9 Possible ameliorating factors of the severity of this disease in the Saudi subjects include continued production of fetal hemoglobin into adult life and/or coexistence of o~- thalassemia. 10.n Deficiency of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) is common in this area and has also been implicated as a possible modifier of sickle cell disease. 12 We have made an attempt to evaluate the biochemical and clinical basis of these interacting factors with sickle cell anemia. Fetal hemoglobin, HbF

Accumulation and salvage of adenosine and inosine by isolated mature cardiac myocytes

Reoxygenation of ischaemic, energy-depleted heart does not result in sufficiently rapid regeneration of normal adenine nucleotide concentrations for preservation of cardiac function and structure. Salvage of nucleoside as a mechanism for restoration of ATP in the post-ischaemic myocardium is limited by efflux of adenosine during ischaemia. Isolated cardiac myocytes have been used to establish the kinetics of uptake and salvage of adenosine and inosine, measuring the distribution of radioactive nucleoside incorporated into ATP, ADP and AMP. Maximum rates of catalysis of reactions on the salvage pathway, and of enzymes competing for substrates on the pathway, have been established in myocyte extracts. Myocytes have little capacity to salvage or catabolise inosine. Enzyme measurements indicate that salvage of adenosine should proceed at 7–8-times the rate exhibited by intact myocytes dependent upon extracellular adenosine as substrate. The data indicate that the rate of transport of adenosine is not determined by its metabolic utilization, but is the rate-limiting step in the salvage of adenosine.

Human adenine phosphoribosyltransferase. Immunochemical quantitation and protein blot analysis of mutant forms of the enzyme.

We have studied the catalytic, immunochemical, and electrophoretic properties of adenine phosphoribosyltransferase in hemolysates from 30 patients with a deficiency of this enzyme in six unrelated families. We have found that: 1) the level of adenine phosphoribosyltransferase enzyme activity and immunoreactive protein in the four homozygous deficient patients was less than 1% of control values; 2) adenine phosphoribosyltransferase enzyme activity was uniformly decreased to approximately 25% of normal in all 26 heterozygotes studied while the level of adenine phosphoribosyltransferase immunoreactive protein was consistent within each kindred but ranged in value from 22% to 112% of control; and 3) protein blot analysis revealed a single isoelectric form of the adenine phosphoribosyltransferase subunit in hemolysate from normal controls and from every heterozygote except for patient M.R. who exhibited both a normal and a more acidic adenine phosphoribosyltransferase subunit species. These studies provide the first evidence for the existence of a variety of different mutations in the structural gene for adenine phosphoribosyltransferase in patients exhibiting a deficiency of enzyme activity. We further conclude from our data that: 1) the variant enzymes are more labile in vivo and/or catalytically nonfunctional, and 2) heterozygotes express only 25% of normal enzyme activity because the normal and variant enzyme subunits form a hybrid dimer which is either more labile or less catalytically active than the normal adenine phosphoribosyltransferase dimer.

Adenine nucleotide levels of canine kidneys during hypothermic aerobic or anaerobic storage in Collins solution.

The influence of retrograde oxygen persufflation (ROP) on renal adenine nucleotide, glucose and lactate contents was examined in canine kidneys during up to 5 days of hypothermic preservation. Organs with or without predamage in 30-min normothermic ischemia were preserved and the results compared to kidneys after hypothermic ischemic storage (HIS): ROP preservation resulted in a significantly higher maintenance of cellular energy-rich substances than in HIS with even normal adenine nucleotide contents and energy charge potentials for the first 24 h. As in HIS preservation of normal kidneys, where the total adenine nucleotide content (SAN) paralleled the loss of viability for the first 1-2 days, in ROP were SAN and even the lactate content also correlated to renal viability for 2-3 days. But in contrast to this correlation, which would favor adenine nucleotide determinations for viability testing, in predamaged kidneys no correlation of SAN or lactate with renal viability was found during HIS or ROP. Thus the determination of cellular energy-rich adenine nucleotides and lactate accumulation confirms the superiority of ROP over HIS preservation. But the SAN or lactate tests lose their prognostic value for posttransplant renal viability if normothermic ischemia precedes the preservation period.

Biogenesis of mitochondria. A requirement for mitochondrial protein synthesis for the formation of a normal adenine nucleotide transporter in yeast mitochondria.

1. Parameters of ATP uptake by fully functional Saccharomyces cerevisiae mitochondria, including kinetic constants, binding constants and sensitivity to atractylate, closely resemble those of mammalian mitochondria. Scatchard plots of atractylate-sensitive adenine nucleotide binding indicate two distinct sites of high affinity (binding constant, K(D)'=1mum), and low affinity (binding constant, K(D)''=20mum) in the ratio 1:3. Uptake has high Arrhenius activation energies (+35 and +57kJ/mol), above and below a transition temperature of 11 degrees C. Atractylate-insensitive ATP uptake is apparently not saturable and has a low Arrhenius activation energy (6kJ/mol), suggesting a non-specific binding process. 2. Kinetic and binding constants for ATP uptake are not significantly changed in catabolite-repressed or anaerobic mitochondrial structures. 3. Inhibition of the mitochondrial protein-synthesizing system by growth of cells in the presence of erythromycin, or loss of mitochondrial DNA by mutation profoundly alters the adenine nucleotide transporter. ATP uptake becomes completely insensitive to atractylate, and the high-affinity binding site is lost. However, the adenine nucleotide transporter does not appear to be totally eliminated, as a moderate amount of saturable low-affinity ATP binding remains. 4. It is concluded that products of the mitochondrial protein-synthesizing system, probably coded by mitochondrial DNA, are required for the normal function of the adenine nucleotide transporter.

De novo synthesis of myocardial adenine nucleotides in the rat. Acceleration during recovery from oxygen deficiency.

De novo synthesis of adenine nucleotides was measured in rat hearts in situ and in isolated perfused rat hearts under normal conditions and during recovery from asphyxia or ischemia. Using l- 14 C-glycine as the precursor substrate, rates of de novo synthesis were determined from the total radioactivity of adenine nucleotides and from the mean specific activity of intracellular glycine. The rate of de novo synthesis of adenine nucleotides was 8.4±1.42 nmoles/g hour -1 in the heart in situ and 1.3±0.12 nmoles/g hour -1 in the isolated perfused heart. De novo synthesis of adenine nucleotides increased almost 100% in the heart in situ and about 580% in the isolated perfused heart during the first hour of recovery from asphyxia or ischemia. This acceleration is regarded as an adaptive process contributing to the postanoxic restoration of normal adenine nucleotide levels. Possible biochemical mechanisms that might be involved in the stimulation of the de novo pathway are a release of feedback inhibition of 5-phosphoribosyl-1-pyrophosphate amidotransferase, an enhanced synthesis of 5-phosphoribosyl-1-pyrophosphate, and an alternate way of 5-phosphoribosyl-amine formation.