The purine nucleotide cycle. Control of phosphofructokinase and glycolytic oscillations in muscle extracts.

Linked oscillations of the glycolytic pathway and the purine nucleotide cycle were studied in particle-free extracts of rat skeletal muscle. Under the conditions used, an accumulation of about 1 muM fructose diphosphate can trigger a sudden increase in phosphofructokinase activity. The activation by fructose diphosphate depends on the presence of AMP. When the AMP concentration drops, phosphofructokinase becomes inhibited, even though the fructose disphosphate concentration remains high. It is concluded that the oscillatory behavior can be of advantage for maintaining a high average [ATP]/[ADP] ratio.     

Oscillations of the glycolytic pathway and the purine nucleotide cycle.

In a treatment modeled after the oscillatory behavior of the glycolytic pathway and the purine nucleotide cycle observed in skeletal muscle extracts, it is shown that the basis of the oscillations is the AMP-dependent activation of phosphofructokinase by fructose diphosphate. Control of phosphofructokinase by the adenine nucleotides alone leads to the establishment of a steady state. Whether steady state or oscillatory behavior occurs depends in part on the activity of glyceraldehyde-3-phosphate dehydrogenase, which controls the rate of removal of fructose diphosphate. Under appropriate conditions oscillatory behavior can maintain a higher [ATP]/[ADP] ratio than steady state behavior. Viewed in the context of conditions that may be encountered in skeletal muscle in vivo, oscillatory behavior of glycolysis is shown to have additional advantages for maintaining a high [ATP]/[ADP] ratio.     

Thermodynamics of the purine nucleotide cycle

Since the standard Gibbs energies of formation are known for all the species in the purine nucleotide cycle at 298.15 K, the functions of pH and ionic strength that yield the standard transformed Gibbs energies of formation of the ten reactants can be calculated. This makes it possible to calculate the standard transformed Gibbs energies of reaction, apparent equilibrium constants, and changes in the binding of hydrogen ions for the three reactions at desired pHs and ionic strengths. These calculations are also made for the net reaction and a reaction that is related to it. The equilibrium concentrations for the cycle are calculated when all the reactants are initially present or only some are present initially. Since the concentrations of GTP, GDP, and P(i) may be in steady states, the equilibrium concentrations are also calculated for the system at specified steady-state concentrations.     

Simulation of the purine nucleotide cycle as an anaplerotic process in skeletal muscle

A computer model of purine nucleotide and citric acid cycles joined through fumarate is given. Steady-state equations corresponding to metabolic enzymes are written based on the information from the literature about their kinetic behavior. Numerical integration of this set of equations is performed and in order to maintain an overall stabilization between the two cycles, enzymatic activities, in the form of V, have been calculated. Sensitivity coefficients for enzymes indicate that the control is exerted, depending upon the intermediate concentrations, and furthermore, it is demonstrated that AMP concentration in muscle should be very low. From stabilization, simulation of exercise conditions has been performed by diminishing [ATP] and increasing accordingly [ADP] and [AMP]. In such conditions the operation of purine nucleotide cycle leads to a considerable increase in the level of citric acid cycle intermediates. Disruption of purine nucleotide cycle by altering some of the three enzymatic steps leads to a lesser increase of these intermediates. The set of results presented seems to confirm the hypothesis that purine nucleotide cycle acts as an anaplerotic process in muscle, as the experimental results of Aragon and Lowenstein (Aragon, J.J., and Lowenstein, J.M. (1980) Eur. J. Biochem. 110, 371-377) suggest.     

Alterations in purine nucleotide metabolism during muscle differentiation in vitro

Pathways of purine nucleotide metabolism affecting the availability of ATP in the muscle tissue were studied in differentiating rat muscle cultures. The rate of de novo purine nucleotide synthesis and of AMP deamination were found to increase markedly with cell differentiation, but the rate of IMP dephosphorylation was similarly low in both myoblasts and contracting fibers. The above differentiation-associated alterations in purine nucleotide metabolism conform with the greater need for ATP as a source of energy in the contracting myotubes.     

A defect of purine nucleotide cycle in the skeletal muscle of hereditary dystrophic mice

The purine nucleotide cycle in the hind leg skeletal muscle of hereditary dystrophic mice ($\text{C57BL}\text{6J}\text{-}\text{dy}\text{dy}$) was investigated. The amount of adenine nucleotide produced from adenylosuccinate by the muscle extract in the dystrophic group was less than 3 % of that in the control group, while adenine nucleotide plus adenylosuccinate converted from IMP in the dystrophic group was about 70 % of that of the control group. Moreover, the activity of AMP deaminase of the dystrophic group was about 50 % of that of the control group. These results indicate that the purine nucleotide cycle is defective in the dystrophic muscle. This abnormality was suggested to be caused by the considerably low activity of adenylosuccinase.     

The purine nucleotide cycle inHelix hepatopancreas

Summary The enzymes adenylosuccinate synthetase (EC 6.3.4.4 IMP: L-aspartate ligase [GDP-forming]), adenylosuccinate lyase (EC 4.3.2.2) and AMP deaminase (EC 3.5.4.6 AMP aminohydrolase) were demonstrated inHelix aspersa hepatopancreas tissue. The presence of these enzymes along with high levels of aspartate transaminase is presumptive evidence for the operation in this tissue of the purine nucleotide cycle. In the absence of evidence that glutamate dehydrogenase acts to release ammonia during amino acid catabolism, it is suggested that the purine nucleotide cycle serves this function. Glutamine synthetase (EC 6.3.1.2 L-glutamate: ammonia ligase [ADP-forming]) was shown to be present primarily in the cytosolic fraction ofHelix hepatopancreas. Since the operation of the purine nucleotide cycle results in the release of ammonia in the cytosol, the localization of glutamine synthetase in this compartment indicates that it is the primary ammonia-detoxifying enzyme and is consistent with the suggestion that the purine nucleotide cycle serves as the major pathway for amino acid catabolism.Supported by grants from the USPHS National Institute of Allergic and Infections Diseases (AI 05006) and the National Science Foundation (PCM-75-13161)     

Metabolic interaction between purine nucleotide cycle and oxypurine cycle during skeletal muscle contraction of different intensities: a biochemical reappraisal

Background

A substrate cycle is a metabolic transformation in which a substrate A is phosphorylated to A?P at the expense of ATP (or another “high energy” compound), and A?P is converted back to A by a nucleotidase or a phosphatase. Many biochemists resisted the idea of such an ATP waste. Why a non-phosphorylated metabolite should be converted into a phosphorylated form, and converted back to its non-phosphorylated form through a “futile cycle”?

Aim of review

In this Review we aim at presenting our present knowledge on the biochemical features underlying the interrelation between the muscle purine nucleotide cycle and the oxypurine cycle, and on the metabolic responses of the two cycles to increasing intensities of muscle contraction.

Key scientific concepts of review

Nowadays it is widely accepted that the substrate cycles regulate many vital functions depending on the expense of large amounts of ATP, including skeletal muscle contraction, so that the expense of some extra ATP and “high energy” compounds, such as GTP and PRPP via substrate cycles, is not surprising. The Review emphasizes the strict metabolic interrelationship between the purine nucleotide cycle and the oxipurine cycle.

     

Determination of metabolite and nucleotide concentrations in proliferating lymphocytes by 1H-NMR of acid extracts

Nuclear magnetic resonance (NMR) studies of extracts have proven to be a powerful window onto the intracellular machinery of cells and tissues. The major advantages of in vitro 1H-NMR, namely chemical preservation, simultaneous detection, identification, and quantitation of compounds, and sensitivity to a large variety of classes of compounds, are employed in this study to characterize the metabolic course of mitogen-stimulated proliferation of human peripheral lymphocytes. A reliable method to quantitate amino acids, metabolic intermediates, soluble membrane lipid precursors, and purine, pyridine and pyrimidine nucleotides is presented, using samples as small as 30 mg wet weight. A total of 53 substances were detected in lymphocytes and other blood cells. During the course of lymphocyte culture, changes in intracellular concentrations of lactate, taurine, inositol and nucleotides, including NAD, IMP and high-energy phosphates, were especially marked. 1H-NMR compares favorably to 31P-NMR and to HPLC, and is especially attractive in light of expectations for future in vivo application.     

The purine nucleotide cycle and ammoniagenesis in rat kidney tubules

The contribution of the purine nucleotide cycle to renal ammoniagenesis was examined in cortical tubule suspensions prepared from acidotic rats and incubated with [alpha-15N]glutamine, [15N]glutamate, or [15N]aspartate. Labeling of ammonia and adenine nucleotides was determined after enzymatic transformations designed to circumvent the technical problem that 15NH3 and H2O have the same nominal mass. Labeling of the adenine nucleotide was undetectable (less than 10%) even after 1 h of incubation. From the measured concentrations of adenine nucleotides and ammonia and the labeling of the ammonia, the flux through the purine nucleotide cycle was calculated to account for less than 1% of the deamination of alpha-amino groups from all three substrates. The glutamate dehydrogenase reaction is therefore the likely pathway for deamination. The rate of 15NH3 production from [alpha-15N]glutamine was two or three times greater than from added [15N]glutamate, indicating a preference for intracellularly generated glutamate. 15NH3 production from added [15N]aspartate was similar to and perhaps slightly greater than that from added [15N]glutamate.     

Vitamin C deficiency activates the purine nucleotide cycle in zebrafish

Vitamin C (ascorbic acid, AA) is a cofactor for many important enzymatic reactions and a powerful antioxidant. AA provides protection against oxidative stress by acting as a scavenger of reactive oxygen species, either directly or indirectly by recycling of the lipid-soluble antioxidant, α-tocopherol (vitamin E). Only a few species, including humans, guinea pigs, and zebrafish, cannot synthesize AA. Using an untargeted metabolomics approach, we examined the effects of α-tocopherol and AA deficiency on the metabolic profiles of adult zebrafish. We found that AA deficiency, compared with subsequent AA repletion, led to oxidative stress (using malondialdehyde production as an index) and to major increases in the metabolites of the purine nucleotide cycle (PNC): IMP, adenylosuccinate, and AMP. The PNC acts as a temporary purine nucleotide reservoir to keep AMP levels low during times of high ATP utilization or impaired oxidative phosphorylation. The PNC promotes ATP regeneration by converting excess AMP into IMP, thereby driving forward the myokinase reaction (2ADP → AMP + ATP). On the basis of this finding, we investigated the activity of AMP deaminase, the enzyme that irreversibly deaminates AMP to form IMP. We found a 47% increase in AMP deaminase activity in the AA-deficient zebrafish, complementary to the 44-fold increase in IMP concentration. These results suggest that vitamin C is crucial for the maintenance of cellular energy metabolism.     

Coordinate enzymatic activity of beta-oxidation and purine nucleotide cycle in a diversity of muscle and other organs of rat.

1. Most mammalian muscles consist of a mixture of different muscle fiber types. 2. We analyzed various muscles with different percentages of slow and fast fibers in addition to other organs of rat for enzyme activities of beta-oxidation and the purine nucleotide cycle (PNC). 3. According to the content of slow-twitch fibers all enzymes of beta-oxidation were high in activity whereas enzymes of the purine nucleotide cycle were low. 4. Amongst all enzymes of beta-oxidation, crotonase showed the highest activity. 5. In heart muscle, enzyme activities of beta-oxidation were even higher than in m. soleus which consists almost exclusively of slow-twitch type I fibers. 6. Measurements of all three enzymes involved in the purine nucleotide cycle revealed high activities in muscles predominantly composed of fast-twitch fibers. 7. It was always adenylate deaminase which revealed the highest activity. 8. Heart muscle showed low activities for enzymes of PNC.     

Characterization of purine nucleotide metabolism in primary rat muscle cultures

The synthesis and metabolic fate of purine nucleotides were studied, employing labeled precursors, in primary rat muscle cultures. The cultures were found to produce purine nucleotides, by de novo and salvage pathways, both exhibiting dependence on cellular availability of substrate 5-phosphoribosyl-1-pyrophosphate (PPRibP). Depletion of cellular PPRibP decelerated the rate of purine synthesis, whereas increasing PPRibP generation by high Pi concentration in the incubation medium, accelerated purine synthesis. Ribose accelerated purine synthesis, indicating that ribose 5-phosphate availability in the cultured muscle is limiting for PPRibP synthesis. The study in the muscle cultures of the metabolic fate if IMP formed from [14C]formate and that of nucleotides formed from labeled purine bases, revealed that the main flow in the nucleotide interconversions pathways is from AMP to IMP. The flow from IMP to GMP and to AMP appeared to be of a lesser magnitude and virtually no flow could be detected from GMP to IMP. The greatest proportion of radioactivity of purine nucleotides following synthesis by either de novo or salvage pathways, accumulated in IMP, reflecting the relative rates of flows between the various nucleotides and probably also a relatively low, or inhibited activity of the IMP nucleotidase. The results suggest that primary muscle cultures are a plausible model for the study of the role of purine metabolism in muscle work.     

Studies of the regulation of purine nucleotide catabolism.

Ehrlich ascites tumor cells containing radioactive ATP were incubated in vitro with a range of concentrations of 2-deoxyglucose in order to produce different rates of ATP catabolism. Concentrations of all radioactive products of ATP catabolism were measured, and apparent rates of adenylate deaminase and inosinate dehydrogenase and of adenylate and inosinate dephosphorylation were calculated. It was concluded that these processes were reggulated primarily by the rate of formation of substrate, and to a lesser extent in some cases, by substrate concentration. No evidence was obtained for regulation of these processes by the concentration of ATP. The deoxyglucose-induced catabolism of radioactive GTP was also studied. When ATP catabolism was induced by incubation with 2,4-dinitrophenol, time courses of accumulation of purine nucleoside monophosphates and rates of alternative pathways of their metabolism were quite different than when deoxyglucose was used.