Regulation of rat heart cytosol 5''-nucleotidase by adenylate energy charge.

A 5'-nucleotidase (EC 3.1.3.5) was highly purified from the soluble fraction of rat heart. The preparation appeared homogeneous by the criterion of polyacrylamide-gel electrophoresis. The enzyme was activated by ATP and ADP, and inhibited by Pi. When AMP was used as substrate, the velocity/substrate-concentration plot was sigmoidal. ATP or ADP changed the plot to hyperbolic and decreased S0.5. Pi increased both the sigmoidicity of the plot and S0.5. When IMP was used as substrate, the velocity/substrate plot was hyperbolic. ATP or ADP decreased Km and increased V. Pi changed the plot to sigmoidal and increased S0.5. Within the range of adenylate energy charge observed in surviving mammalian cells (0.7-0.9), the rate of AMP-hydrolysing activity catalysed by the 5'-nucleotidase increased sharply with decreasing energy charge. The highest activity was observed at an energy-charge value of about 0.6. The response was also observed in the presence of Pi. No change in IMP-hydrolysing activity was observed in the physiological range of adenylate energy charge, but in the presence of Pi the activity gradually increased with increasing energy charge. These results suggest the possibility that this enzyme participates in production of adenosine, a vasodilator, during hypoxia and in removal of IMP, which accumulates during the hypoxia, in the heart.     

AMP deaminase from baker's yeast. Purification and some regulatory properties.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.     

Aspects of purine metabolism in the gill epithelium of rainbow trout, Salmo gairdneri Richardson.

1. Enzymes interconnecting the adenylate pool were present in high concentration. 2. AMP and adenosine were easily deaminated by the corresponding enzymes whose high levels were detected. 3. Adenylate was hydrolyzed either by deamination to yield IMP which was further dephosphorylated to inosine or by dephosphorylation to adenosine followed by deamination to inosine. 4. Incubation of gill extract with [-14C]-AMP in the presence and absence of ATP but with adenosine deaminase inhibitors allowed demonstration that ATP controlled the balance between these pathways. 5. Some biochemical properties of 5'-nucleotidase. AMP deaminase and adenosine deaminase were defined. 6. Purine salvage enzymes were also estimated.     

The proposed 5'-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5'-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577-584) mentioned that this phenomenon resulted from the presence of a 5'-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5'-[3H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [3H]Adenosine derived from 5'-[3H]AMP hydrolysis was further transformed into [3H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [3H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5'-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5'-nucleotidase inhibitor. We did not observe 5'-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.     

Major determinants of purine excretion from human lymphoblasts

WI-L2 B lymphoblasts deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) excreted amounts of hypoxanthine two to three times larger than CEM T lymphoblasts deficient in HGPRT, despite similar growth rates. ATP consumption occurred at a higher rate in WI-L2 cells than in CEM cells when cultivated in a glucose-free buffer, because of higher RNA synthesis in WI-L2 cells. The introduction of actinomycin D and azaserine resulted in lower hypoxanthine excretion in WI-L2 cells than in CEM cells, not in parallel with changes of the adenylate pool size. When the energy charge was high, de novo purine synthesis was a major determinant for purine excretion. The adenylate pool ratio (AMP/ATP) change caused by the introduction of oligomycin was greater during ATP depletion and recovery in WI-L2 cells than in CEM cells. WI-L2 cells were observed to have AMP deaminase activity three to four times higher than CEM cells. The major component of AMP deaminase in these cells was liver type. The higher rate of RNA synthesis caused greater changes of (AMP/ATP) and required higher AMP deaminase activity for recovery. When the energy charge was low, AMP deaminase was a major determinant for purine excretion.     

The regulatory role of spermine and fatty acid in the interaction of AMP deaminase with phosphofructokinase

The role of fatty acid and polyamine in the interaction of AMP deaminase (EC 3.5.4.6)-ammonium system with glycolysis was investigated using permeabilized yeast cells. (1) The addition of fatty acid inhibited the activity of AMP deaminase in situ, resulting in a decrease in the total adenylate pool depletion, and in the recovery of the adenylate energy charge. (2) The addition of fatty acid resulted in an indirect decrease in the activity of phosphofructokinase (EC 2.7.1.11) through a reduced level of ammonium ion; fatty acid itself did not inhibit phosphofructokinase activity in the presence of excess ammonium ion. (3) Spermine protected AMP deaminase from inhibition by fatty acid: the increased ammonium level enhanced phosphofructokinase activity, glycolytic flux and the recovery of the energy charge. In contrast, alkali metals, which are also activators of AMP deaminase had little effect on the inhibition of the enzyme. The inhibition of glycolysis by fatty acid and its reversal by polyamine can be accounted for by the changes in ammonium ion through the action of AMP deaminase-ammonium system, and the physiological relevance is discussed.     

In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts

The role of AMP deaminase reaction in the stabilization of the adenylate energy charge was investigated using permeabilized yeast cells. The addition of P_i or Zn²⁺, which inhibits AMP deaminase, remarkably retarded the depletion of total adenylate pool and the recovery of the adenylate energy charge. Polyamine, an activator of the enzyme, decreased total adenylates, resulting in the enhanced recovery of the energy charge in situ. AMP deaminase can act as a regulatory enzyme in the system that stabilizes the adenylate energy charge in yeast cells under the conditions of severe metabolic stress.     

Activities of adenylate-degrading enzymes in muscles from vertebrates and invertebrates

Activities of adenylate-degrading enzymes in muscles of vertebrates and invertebrates were determined. Mammalian and fish muscles showed a markedly higher activity of AMP deaminase with a lower level of adenosine deaminase and 5'-nucleotidase. Cephalopods showed an active adenosine deaminase and a 5'-nucleotidase which preferred AMP as the substrate. Negligible deamination of AMP and adenosine and little phosphohydrolase activity toward AMP and IMP were observed in the shellfish muscles. Adenine nucleotides can be degraded to form IMP via the AMP deaminase reaction in vertebrate muscles, while dephosphorylation of AMP to adenosine, which is then converted to inosine, appears to proceed in cephalopods. Adenylates can be hardly degraded in shellfish muscles.     

The role of adenosine monophosphate nucleosidase in the regulation of adenine nucleotide levels in Azotobacter vinelandii during aerobic-anaerobic transitions

When cultures of Azotobacter vinelandii are made anaerobic the adenylate pool size remains constant or increases slightly while the adenylate energy charge decreases. Under these conditions, cell growth stops but the cells remain viable for at least 5 h with the decreased energy charge. The changes in the adenylate pool during the aerobic-anaerobic transition include: the formation of adenylates as a result of RNA degradation; the degradation of a portion of the excess AMP to form hypoxanthine by the sequential actions of AMP nucleosidase and adenine deaminase; an increase in the total adenylate pool which is stabilized at approximately 1.5 times the level in growing cells; and stabilization of the adenylate energy charge at a value near 0.3. The degradation of AMP is regulated by AMP nucleosidase, an allosteric enzyme which is activated by MgATP^2? and inhibited by P_i. The in vivo activity of AMP nucleosidase was estimated by measuring the rate of hypoxanthine formation in the culture or by measuring the activity of purified enzyme at the concentrations of AMP, ATP, and P_i found in the cells. The maximum estimated in vivo rate of AMP degradation was less than 3% of the catalytic capacity of AMP nucleosidase. Thus ample activity is present for rapid adjustments of the AMP levels in these cells. Expression of AMP nucleosidase catalytic activity is tightly controlled since high constant concentrations of intracellular AMP can be maintained for extended time periods at low adenylate energy charge values. Under these conditions controlled degradation of AMP can occur to maintain a constant AMP concentration.     

Effects of sterol manipulation on microsomal desaturase activities in Tetrahymena: with regard to thermal acclimation

The role of NH+4 ion and AMP deaminase reaction in the activation of phosphofructokinase with respect to its response to the adenylate energy charge was investigated using permeabilized yeast cells. (a) Phosphofructokinase and AMP deaminase were activated by the decrease in the adenylate energy charge. The addition of NH+4 further stimulated the phosphofructokinase activity in the presence of intracellular level of K+, and the optimal energy charge value giving the maximal response of the enzyme was shifted from 0.3 to the value above 0.5. (b) The increase in NH+4 ion produced through the activation of AMP deaminase by spermine which shows no direct action on the phosphofructokinase activity can activate phosphofructokinase with shift of the optimal energy charge value of the enzyme to 0.5 in the presence of K+, whereas the optimal energy charge value for AMP deaminase reaction was not affected by the addition of spermine. Phosphofructokinase can be activated most effectively by the physiological decrease in the energy charge under the condition of increased NH+4 in the presence of K+. The possibility that the interaction of phosphofructokinase with AMP deaminase under hypoxic condition might be a contributing factor to the Pasteur effect is discussed.     

Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation.

Adenine nucleotide breakdown to nucleosides and purine bases was measured in cultures of human lymphoblastoid cells following: 1) the inhibition of oxidative phosphorylation in the absence of glucose or 2) the addition of 2-deoxyglucose. A mutant cell line, deficient in adenosine kinase, in the presence of an adenosine deaminase inhibitor was used to measure utilization of the two pathways of AMP catabolism involving initial action of either purine 5'-nucleotidase or AMP deaminase. In such a system the appearance of adenosine induced by the oxidative phosphorylation inhibitor, rotenone, implies that approximately 70% of AMP breakdown occurs via dephosphorylation. By the same method, deamination accounts for 82% of AMP breakdown when 2-deoxyglucose is added. The occurrence of AMP dephosphorylation is not correlated with elevated concentrations of substrate or with decreased concentrations of the inhibitors of 5'-nucleotidase, ATP and ADP. Dephosphorylation occurs if, and only if, the adenylate energy charge decreases to about 0.6 in these experiments. In cultures deprived of glucose and oxygen, adenine nucleotide degradation via dephosphorylation results in recovery of normal energy charge values.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Role of adenylate kinase, AMP deaminase and 5'-nucleotidase in the metabolism of adenylic nucleotides

The activities of adenylate kinase, AMP-deaminase and 5'-nucleotidase in various tissues of the rat were studied. The activity of the forward adenylate kinase reaction (ATP + AMP----2 ADP) against the back one (2 ADP----ATP + AMP) was predominant. The liver was shown to contain two, while the blood serum--three adenylate kinase isoenzymes. In the skeletal muscles, the catabolism of adenylic acid involving AMP-deaminase and 5'-nucleotidase predominantly occurred via deamination, in the liver--via dephosphorylation, while in the leucocytes, erythrocytes and blood serum the activity of these processes was essentially the same. In vitro, ATP enhanced the activity of AMP-deaminase in the liver, leucocytes and erythrocytes and decreased it in the blood serum. Under effects of ATP, the activity of 5'-nucleotidase in the leucocytes and blood serum was markedly elevated, that in the liver and erythrocytes was unaffected.     

Induction and repression of enzymes involved in exogenous purine compound utilization of Bacillus cereus

5'-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine, nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented that during growth of B. cereus in the presence of AMP, the concerted action of 5'-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B. cereus acts as a translocase of the ribose moiety of inosine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol Chem. 253, 7905-7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Adenosine-5-monophosphate catabolism in frog liver

1. AMP catabolism in frog liver extract was found to proceed exclusively through the formation of IMP. Further metabolism of IMP is relatively slow. 2. Among the enzymes involved in AMP catabolism, AMP deaminase is most active and adenosine deaminase and AMP 5'-nucleotidase exhibit only 20 and 10% of AMP deaminase activity respectively.     

Stabilization of the adenylate energy charge in erythrocytes of rats and humans at high altitude hypoxia.

1. Stabilization of adenylate energy charge and control of adenylate pool were analysed in the erythrocytes of the rat and the human exposed to highly hypoxic conditions. 2. Red cell energy charge was decreased in the rats exposed to a simulated altitude of 5000-8000 m, and then recovered to the normal value with the depletion of adenylate pool. 3. The energy charge and the adenylate pool size of the human erythrocytes did not show any change under highly hypoxic conditions. 4. Anaerobic incubation of rat erythrocytes caused a marked decrease in the energy charge, and its recovery was accompanied by the depletion of total adenylates. 5. The energy charge and total adenylates of human red cells did not change under the anaerobic incubation of erythrocytes. 6. These results suggest that the energy charge of rat erythrocytes can be controlled by depletion of the adenylate pool, but the adenylate degradation is not responsible for the stabilization of the energy charge in human erythrocytes.     

AMP deaminase reaction as a control system of glycolysis in yeast. Role of ammonium ion in the interaction of phosphofructokinase and pyruvate kinase activity with the adenylate energy charge

The role of ammonium ion and AMP deaminase (EC 3.5.4.6) reaction in the activation of phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) by the decrease in the adenylate energy charge was investigated using permeabilized yeast cells. Response of AMP deaminase, phosphofructokinase, and pyruvate kinase to variation in the energy charge is typical of the ATP-regenerating enzymes: an activation with the decrease in the energy charge under the in situ conditions. The addition of polyamine activated AMP deaminase in situ, resulting in the subsequent increase in ammonium production, which can stimulate the phosphofructokinase activity with the increase in the optimal energy charge value giving maximal activity of the enzyme. The optimal energy charge value of phosphofructokinase was 0.2-0.25 in the absence of ammonium ion and was shifted to the value above 0.5 by the addition of ammonium ion, whereas Pi, an activator of the enzyme showed little effect on the increase in the optimal energy charge value. The optimal energy charge value of AMP deaminase and pyruvate kinase was not affected by the addition of their effectors. Modulation of the response to the energy charge of phosphofructokinase and pyruvate kinase was analyzed in terms of the "activation coefficient," which was defined as the ratio of the activity at the energy charge of 0.6 to that at the value of 0.9. Activation of phosphofructokinase by the physiological decrease in the energy charge (0.9 to 0.6) can be enhanced by the increase in ammonium ion specifically, although the coefficient of pyruvate kinase remained unaffected by ammonium ion. These results suggest that the AMP deaminase reaction as an ammonium-forming reaction can participate in a key role in the stimulation of phosphofructokinase or glycolytic flux in cells.     

AMP deaminase as a control system of glycolysis in yeast. Mechanism of the inhibition of glycolysis by fatty acid and citrate

The role of fatty acid and citrate on the interaction of the AMP deaminase (EC 3.5.4.6) reaction with glycolysis was investigated using permeabilized yeast cells. (a) Linolenate and citrate inhibited glycolytic flux and the recovery of the adenylate energy charge; however, linolenate remarkably retarded the depletion of the total adenylate pool, which was not at all affected by the addition of citrate. (b) Linolenate inhibited AMP deaminase activity in situ, resulting in the subsequent decrease in ammonium production, which reduced the activity of 6-phosphofructokinase (EC 2.7.1.11), whereas linolenate itself had no ability to inhibit the phosphofructokinase activity in the presence of excess ammonium concentration. (c) Citrate inhibited the activity of phosphofructokinase in situ in the presence and absence of ammonium ion, followed by an inhibition of glycolysis; however, AMP deaminase activity was not inhibited by citrate. The inhibition of glycolysis by fatty acids can be accounted for by the lowered activity of phosphofructokinase as a result of the decreased level of ammonium ion through the inhibition of the AMP deaminase reaction by these ligands, whereas the effect of citrate on glycolysis is a direct inhibition of phosphofructokinase without affecting the activity of AMP deaminase. Fatty acid and citrate, a principal metabolic product of fatty acid oxidation, can be responsible for the control of glycolysis in two different manners.     

Developmental Changes in the Activity of Enzymes of Purine Metabolism in Rat Neuronal Cells in Culture and in Whole Brain

The activities (Vmax) of several enzymes of purine nucleotide metabolism were assayed in premature and mature primary rat neuronal cultures and in whole rat brains. In the neuronal cultures, representing 90% pure neurons, maturation (up to 14 days in culture) resulted in an increase in the activities of guanine deaminase (guanase), purine-nucleoside phosphorylase (PNP), IMP 5'-nucleotidase, adenine phosphoribosyltransferase (APRT), and AMP deaminase, but in no change in the activities of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenosine deaminase, adenosine kinase, and AMP 5'-nucleotidase. In whole brains in vivo, maturation (from 18 days of gestation to 14 days post partum) was associated with an increase in the activities of guanase, PNP, IMP 5'-nucleotidase, AMP deaminase, and HGPRT, a decrease in the activities of adenosine deaminase and IMP dehydrogenase, and no change in the activities of APRT, AMP 5'-nucleotidase, and adenosine kinase. The profound changes in purine metabolism, which occur with maturation of the neuronal cells in primary cultures in vitro and in whole brains in vivo, create an advantage for AMP degradation by deamination, rather than by dephosphorylation, and for guanine degradation to xanthine over its reutilization for synthesis of GMP. The physiological meaning of the maturational increase in these two ammonia-producing enzymes in the brain is not yet clear. The striking similarity in the alterations of enzyme activities in the two systems indicates that the primary culture system may serve as an appropriate model for the study of purine metabolism in brain.