Properties of adenosine monophosphate deaminase of Candida albicans.

Adenosine monophosphate deaminase (AMPD; EC 3.5.4.6) catalyses the hydrolysis of adenosine monophosphate (AMP) to commensurate amounts of inosine monophosphate (IMP) and ammonia. The production of AMP deaminase in Candida albicans was measured in Lee's medium grown cultures. The highest AMPD activity was observed at 24 h of growth. The enzyme had an optimum pH and temperature at 6-7 and 28 degrees C, respectively. This enzyme was inhibited under iron-limited growth conditions as well as by protease inhibitors. The AMPD of C. albicans showed a moderate increase in activity when cultures were grown in the presence of the divalent cations Mg2+, Ca2+, and Zn2+. Moreover, ADP, ATP, adenine, adenosine, deoxyribose and hypoxanthine increased the enzyme activity. Cultures grown in trypticase soy broth exhibited maximum AMPD activity compared with those grown in Sabouraud dextrose broth or Lee's medium.     

Folic acid deaminase activity during development in Dictyostelium discoideum.

Folic acid attracts vegetative amoebae of Dictyostelium discoideum. Secreted by bacteria, it may act as a food-seeking device. The inactivation of this attractant is catalyzed by a deaminase. As assay has been developed to measure the folic acid deaminase activity. In addition to cell-surface an intracellular deaminase, the amoebae of D. discoideum release the enzyme into the medium. The pH optimum of the extracellular enzyme was 6.0, and higher for the cell-associated deaminases. The extracellular enzyme was secreted maximally by vegetative amoebae, and its activity diminished during cell differentiation. The cell-surface bound enzyme was less active than the extracellular enzyme, and its activity decreased twofold during a 6-h starvation period. The enzyme activity of homogenates and 48,000 x g pellets diminished during this period 35 to 40%. The supernatant of a homogenate had a higher deaminase activity than the homogenate itself or its pellet; this suggests the presence of an inhibitor in the particulate fraction. The underlying mechanism for inactivation of folic acid has similar characteristics as that for inactivation of cyclic adenosine monophosphate.     

AMP deaminase in Dictycostelium discoideum: Increase in activity following nutrient deprivation induced by starvation or hadacidin

Summary AMP deaminase, the activity that catalyzes the deamination of AMP to form IMP and NH₃ has been measured in Dictyostelium discoideum. A new procedure to assay the activity of this enzyme was developed using formycin 5-monophosphate, a fluorescent analog of AMP as the substrate, and ionpaired reverse phase HPLC to separate the reactants and products. Quantitation of the formycin containing compounds was accomplished at 290 nm. At this wavelength adenosine containing compounds were not detected and activity could be monitored in the presence of its activator ATP. The AMP deaminase activity in vegetative cells was 7.4 nmols/min/mg proteins while the activity in cells measured at 2 and 6 hrs after starvation-induced growth-arrest was 376 nmols/min/mg protein... a 51-fold increase. When vegetative cells were treated with hadacidin, a drug that restricts de novo AMP synthesis and pinocytosis, the activity of the AMP deaminase was 511 nmols/min/mg protein... a 70-fold increase compared to that in untreated vegetative cells. Smaller increases were noted following the inhibition of growth with the drugs cerulenin and vinblastine, as well as after the inhibition of de novo GMP synthesis with the drug mycophenolic acid or the partial inhibition of de novo AMP synthesis with analogs of hadacidin, N-hydroxyglycine and N-formylglycine. In addition, when the activity of two other enzymes involved in purine metabolism, namely adenosine kinase and hypoxanthine-guanine phosphoribosyl transferase, was measured in vegetative cells, and the activity of both compared to that measured in starvation and hadacidin induced growth-arrested cells, showed no significant changes. These data suggest that the changes in the activity of the AMP deaminase are in response to nutrient deprivation and further, that as a consequence of the increase in AMP deaminase activity, ammonia will be produced and an increase in pH should follow. The production of ammonia and its effect on development implicates the AMP deaminase in the early differentiation of this organism.     

Selective adenosine release from human B but not T lymphoid cell line

Intracellular adenosine formation and release to extracellular space was studied in WI-L2-B and SupT1-T lymphoblasts under conditions which induce or do not induce ATP catabolism. Under induced conditions, B lymphoblasts but not T lymphoblasts, release significant amounts of adenosine, which are markedly elevated by adenosine deaminase inhibitors. In T lymphoblasts, under induced conditions, only simultaneous inhibition of both adenosine deaminase activity and adenosine kinase activities resulted in small amounts of adenosine release. Under noninduced conditions, neither B nor T lymphoblasts release adenosine, even in the presence of both adenosine deaminase or adenosine kinase inhibitors. Comparison of B and T cell's enzyme activities involved in adenosine metabolism showed similar activity of AMP deaminase, but the activities of AMP-5'-nucleotidase, adenosine kinase and adenosine deaminase differ significantly. B lymphoblasts release adenosine because of their combination of enzyme activities which produce or utilize adenosine (high AMP-5'-nucleotidase and relatively low adenosine kinase and adenosine deaminase activities). Accelerated ATP degradation in B lymphoblasts proceeds not only via AMP deamination, but also via AMP dephosphorylation into adenosine but its less efficient intracellular utilization results in the release of adenosine from these cells. In contrast, T lymphoblasts release far less adenosine, because they contain relatively low AMP-5'-nucleotidase and high adenosine kinase and adenosine deaminase activities. In T lymphoblasts, AMP formed during ATP degradation is not readily dephosphorylated to adenosine but mainly deaminated to IMP by AMP deaminase. Any adenosine formed intracellularly in T lymphoblasts is likely to be efficiently salvaged back to AMP by an active adenosine kinase. In general, these results may suggest that adenosine can be produced only by selective cells (adenosine producers) whereas other cells with enzyme combination similar to SupT1-T lymphoblasts can not produce significant amounts of adenosine even in stress conditions.     

Comparative enzymology of AMP deaminase,adenylate kinase,and creatine kinase in vertebrate heart and skeletal muscle: the characteristic AMP deaminase levels of skeletal versus cardiac muscle are reversed in the North American toad

The specific activity of three characteristic enzymes, adenylate deaminase, adenylate kinase, and creatine kinase, in the skeletal muscles and heart of a variety of vertebrate land animals, including the human, are surveyed. Data from this study and available studies in the literature suggest that adenosine monophosphate deaminase in land vertebrates is quite high in white skeletal muscle, usually somewhat lower in red muscle, and 15-to 500-fold lower in cardiac muscle. Adenosine monophosphate deaminase is active primarily under ischemic or hypoxic conditions which occur frequently in white muscle, only occasionally in red muscle, and ought never occur in heart muscle, and this may therefore account for observed enzyme levels. The common North American toad, Bufo americanus, provides a striking exception to the rule with cardiac adenosine monophosphate deaminase as high as in mammalian skeletal muscle, whereas its skeletal muscle level of adenosine monophosphate deaminase is several times lower. The exceptional levels in the toad are not due to a change in substrate binding and are not accompanied by comparable change in the level of adenylate or creatine kinase. Nor do they signal any major change in isozyme composition, since a human muscle adenosine monophosphate deaminase-specific antiserum reacts with toad muscle adenosine monophosphate deaminase, but not with toad heart adenosine monophosphate deaminase. They do not represent any general anuran evolutionary strategy, since the bullfrog (Rana catesbeiana) and the giant tropic toad (Bufo marinus) have the usual vertebrate pattern of adenosine monophosphate deaminase distribution. Lower skeletal muscle activities in anurans may simply represent the contribution of tonic muscle fiber bundles containing low levels of adenosine monophosphate deaminase, but the explanation for the extremely high adenosine monophosphate deaminase levels in heart ventricular muscle is not apparent.Abbreviations AK adenylate kinase - AMP adenosine monophosphate - AMPD, AMP deaminase - CPK creatine (phospho)kinase - EHNA erythro-9-(2-hydroxy-3-nonyl)-adenine-HCl     

Flow system for fish freshness determination based on double multi-enzyme reactor electrodes

A double reactor system for the determination of fish and shellfish freshness using the freshness indicator, K-value (K=[(HxR+Hx)/(ATP+ADP+AMP+IMP+HxR+Hx)]x100), was developed, where ATP, ADP, AMP, IMP, HxR and Hx are adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, inosine and hypoxanthine, respectively. The system consisted of a pair of enzyme reactors with an oxygen electrode positioned close to the respective reactor. The enzyme reactor (I) was packed with nucleoside phosphorylase and xanthine oxidase immobilized simultaneously on chitosan beads (immobilized enzyme A). Similarly, the enzyme reactor (II) was packed with immobilized enzyme A and immobilized enzyme B (co-immobilized alkaline phosphatase and adenosine deaminase). Moreover, this reactor consisted of two layers, the enzyme A and enzyme B (1:1). A good correlation was obtained between K values, which were determination by the proposed system and by the HPLC method. One assay could be completed within 5 min. The signal for the determination of K value of fish and shellfish was reproducible within 2.3%. The long-term stability of the enzyme reactors was evaluated at 30 degrees C for 28 days.     

A specific AMP deaminase assay and its application to tissue homogenates

1. A rapid method for the determination of AMP and IMP by HPLC is described. 2. Its application to the assay of AMP deaminase allows the specific determination of enzyme activities in crude extracts, eliminating any interference by other enzyme systems (5'-nucleotidase and adenosine deaminase). 3. The method was routinely used for the determination of the AMP deaminase activity in the muscles of marine animals.     

Interactions between catecholamines,methyl xanthines and adenosine in regulation of cyclic AMP accumulation in hamster adipocytes

In the presence of either methyl xanthines or adenosine deaminase, isoproterenol elicited large dramatic increases in accumulation of cyclic AMP. In contrast, cyclic AMP accumulation in response to epinephrine or norepinephrine was not potentiated by either methyl xanthines or by adenosine deaminase. Blocking the alpha adrenergic activity of norepinephrine and epinephrine with phentolamine established synergism between these catecholamines and methyl xanthines and adenosine deaminase. The activity of the particulate phosphodiesterase was not influenced by norepinephrine suggesting that the lack of synergism between the catecholamines norepinephrine and epinephrine and methyl xanthines is unrelated to this enzyme. The data are interpreted to suggest that the alpha adrenergic activity of catecholamines prevents the potentiation of cyclic AMP accumulation that occurs when the action of endogenously produced adenosine is interfered with, either by its degradation with adenosine deaminase or by receptor blockade with methyl xanthine. Because a major action of adenosine on fat cells is to inhibit adenylate cyclase it is suggested that alpha adrenergic receptor activation limits the extent to which the enzyme adenylate cyclase can be activated in a fashion similar to that of adenosine.     

Cell-free synthesis of active ribulose-1,5-bisphosphate carboxylase in the mesophyll chloroplasts of Sorghum vulgare

In the presence of either methyl xanthines or adenosine deaminase, isoproterenol elicited large dramatic increases in accumulation of cyclic AMPP. In contrast, cyclic AMP accumulation in response to epinephrine or norepinephrine was not potentiated by either methyl xanthines or by adenosine deaminase. Blocking the alpha adrenergic activity of norepinephrine and epinephrine with phentolamine established synergism between these catecholamines and methyl xanthines and adenosine deaminase. The activity of the particulate phosphodiesterase was not influenced by norepinephrine suggesting that the lack of synergism between the catecholamines norepinephrine and epinephrine and methyl xanthines is unrelated to this enzyme. The data are interpreted to suggest that the alpha adrenergic activity of catecholamines prevents the potentiation of cyclic AMP accumulation that occurs when the action of endogenously produced adenosine is interfered with, either by its degradation with adenosine deaminase or by receptor blockade with methyl xanthine. Because a major action of adenosine on fat cells is to inhibit adenylate cyclase it is suggested that alpha adrenergic receptor activation limits the extent to which the enzyme adenylate cyclase can be activated in a fashion similar to that of adenosine.     

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.     

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaryotic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing paired-ion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formycin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent Km of 18.2 microM and an apparent Vmax of 32.4 mmol min-1 mg-1 were observed for the activity. A purine-nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent Km of 236 microM and Vmax of 2.78 mumol min-1 mg-1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent Ki value of 136 microM was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a Km value for GTP, IMP, and aspartic acid of 23, 34, and 714 microM, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have Km and Vmax values for hypoxanthine of 55.5 microM and 34.3 nmol-1 min-1 mg-1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of purine nucleotide metabolism in primary rat cardiomyocyte cultures

Primary rat cardiomyocyte cultures were utilized as a model for the study of purine nucleotide metabolism in the heart muscle, especially in connection with the mechanisms operating for the conservation of adenine nucleotides. The cultures exhibited capacity to produce purine nucleotides from nonpurine molecules (de novo synthesis), as well as from preformed purines (salvage synthesis). The conversion of adenosine to AMP, catalyzed by adenosine kinase, appears to be the most important physiological salvage pathway of adenine nucleotide synthesis in the cardiomyocytes. The study of the metabolic fate of IMP formed from [14C]formate or [14C]hypoxanthine and that of AMP formed from [14C]adenine or [14C]adenosine revealed that in the cardiomyocyte the main flow in the nucleotide interconversion pathways is from IMP to AMP, whereas the flux from AMP to IMP appeared to be markedly slower. Following synthesis from labeled precursors by either de novo or salvage pathways, most of the radioactivity in purine nucleotides accumulated in adenine nucleotides, and only a small proportion of it resided in IMP. The results suggest that the main pathway of AMP degradation in the cardiomyocyte proceeds through adenosine rather than through IMP. About 90% of the total radioactivity in purines effluxed from the cells during de novo synthesis from [14C]formate or following prelabeling of adenine nucleotides with [14C]adenine were found to reside in hypoxanthine. The activities in cell extracts of AMP 5'-nucleotidase and IMP 5'-nucleotidase, which catalyze nucleotide degradation, and of AMP deaminase, a key enzyme in the purine nucleotide cycle, were low. The nucleotidase activity resembles, and that of the AMP deaminase contrasts the respective enzyme activities in extracts of cultured skeletal-muscle myotubes. The results indicate that in the cardiomyocyte, in contrast to the myotube, the main mechanism operating for conservation of nucleotides is prompt phosphorylation of AMP, rather than operation of the purine nucleotide cycle. The primary cardiomyocyte cultures are a plausible model for the study of purine nucleotide metabolism in the heart muscle.     

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.     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

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.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.     

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.     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [14C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2'-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined in cell extracts. The results demonstrate that under physiological conditions, there is a small but significant flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5'-nucleotidase (EC 3.1.3.5), reflecting the markedly higher Vmax/Km ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher Vmax/Km ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

The conversion of ATP to IMP by muscle surface enzymes

These experiments showed that an isolated muscle bundle could be used to study, simultaneously, ion transport and the activity of surface enzymes. Frog muscles were carefully dissected and incubated for four hours in Ringer's solution containing a tris buffer and 3 mM ATP; at various times samples of the medium were chromatographed and analysed spectrophotometrically for nucleotide content. Samples of the final medium were analysed for inorganic phoshpate and for ammonia. The results demonstrated the conversion of adenosine triphosphate to inosine monophosphate, via adenosine diphosphate and adenosine monophosphate. Under the conditions of the experiment IMP was detected on chromatograms within 20 minutes of incubation; at the end of four hours the media had concentrations of 2.3 mM IMP, 4.4 mM inorganic phosphate and 2.6 mM ammonia, showing a stoichiometric relation among the products formed. There was convincing evidence that the enzymes involved (ATPase, adenylate kinase and AMP deaminase) must be situated close to or on the muscle surface. No effect of ouabain (1 μM) on the activity of the ATPase, adenylate kinase or deaminase could be found in these experiments, but the drug inhibited Na and K recovery from a Na-loaded, K-depleted state.     

Inhibition of 5′-nucleotidase from Ehrlich ascites-tumour cells by nucleoside triphosphates

1. 5'-Nucleotidase activity was obtained in a soluble form after treatment of a particulate fraction from Ehrlich ascites-tumour cells with deoxycholate. The relative rates of hydrolysis of 6-thioinosine 5'-phosphate, UMP, AMP, CMP, GMP, IMP, xanthosine monophosphate, thymidine monophosphate and 2',3'-AMP were 180, 129, 100, 93, 83, 79, 46, 41 and 3 respectively. 2. Values found for the Michaelis constant were: AMP, 67+/-12mum; IMP, 111+/-8mum; GMP, 93mum. 3. ATP and thymidine triphosphate were competitive inhibitors of AMP hydrolysis (inhibitor constants 0.4 and 4.8mum respectively); UTP, GTP and CTP were mixed competitive and non-competitive inhibitors. Thymidine triphosphate was a competitive inhibitor of IMP hydrolysis (inhibitor constant 14.4mum) and ATP, UTP and GTP showed mixed competitive and non-competitive inhibition. 4. ATP, thymidine triphosphate, UTP, GTP and CTP did not completely inhibit hydrolysis of AMP, IMP and UMP; the concentrations of ATP required to inhibit AMP and IMP hydrolysis by 50% were 12 and 230mum respectively. 5. Non-hyperbolic curves relating activity to UMP concentration were obtained in the presence and absence of triphosphates. 6. After fractionation on Sephadex G-200 columns a single peak of 5'-nucleotidase activity (particle weight 120000-125000) was obtained with AMP, IMP and GMP as substrates. UMP hydrolysis was catalysed by enzyme in this peak and in two slower peaks corresponding to apparent particle weights of 32000 and 16000; a single component (particle weight 120000), reacting with UMP and insensitive to UTP inhibition, was obtained when the column was eluted with buffer containing 1mm-UMP. 7. The possible significance of the results in the regulation of tumour-cell 5'-nucleotidase is discussed.