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PubMed2003

Dominant localization of adenosine deaminase in leptomeninges and involvement of the enzyme in sleep.

Adenosine is an endogenous hypnotic molecule. However, the mechanism by which the level of extracellular adenosine is regulated remains to be elucidated. We found by Northern hybridization and enzyme assay that ecto-5(')-nucleotidase and adenosine deaminase (ADA), major enzymes responsible for the production and degradation of adenosine, respectively, were localized most abundantly in the leptomeninges within the rat brain. Immunohistochemical study showed that ADA was dominantly localized in arachnoid barrier and trabecular cells of the leptomeninges. In vivo microdialysis demonstrated that externally applied adenosine was rapidly metabolized by ADA to inosine in the subarachnoid space. Perfusion of an ADA inhibitor, coformycin, increased the extracellular adenosine level in the subarachnoid space under the rostral basal forebrain. When coformycin was continuously infused into the subarachnoid space, non-rapid eye movement sleep was increased with prolonged duration of the sleep episode. These results demonstrate that the leptomeninges control the extracellular level of adenosine in the subarachnoid space by their high 5(')-nucleotidase and ADA activities and regulate non-rapid eye movement sleep.

Adenosine↗
PubMed1986

Synthesis and biochemical properties of 8-amino-6-fluoro-9-beta-D-ribofuranosyl-9H-purine.

The synthesis and characterization of 8-amino-6-fluoro-9-beta-D-ribofuranosyl-9H-purine (3a) are presented. This compound is a substrate for adenosine deaminase and adenosine kinase. In L1210 cells 3a is converted to 8-aminoinosine monophosphate (4b), apparently by the action of AMP deaminase on the monophosphate of 3a, as well as to the triphosphate derivative of 3a. Pentostatin was used to inhibit adenosine deaminase, and coformycin was used to inhibit AMP deaminase in experiments designed to delineate the metabolic fate of 3a. Pentostatin was without influence on the cytotoxicity of 3a, but coformycin potentiated the cytotoxicity. The potentiation was associated with an increased cellular concentration of phosphates of 3a and a decreased concentration of 4b.

AMP Deaminase↗
PubMedDate not supplied

L-nucleoside analogues as potential antimalarials that selectively target Plasmodium falciparum adenosine deaminase.

The L-stereoisomer analogues of D-coformycin selectively inhibited P. falciparum adenosine deaminase (ADA) in the picomolar range (L-isocoformycin, Ki 7 pM; L-coformycin, Ki 250 pM). While the L-nucleoside analogues, L-adenosine, 2,6-diamino-9-(L-ribofuranosyl)purine and 4-amino-1-(L-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine were selectively deaminated by P. falciparum ADA, L-thioinosine and L-thioguanosine were not. This is the first example of 'non-physiological' L-nucleosides that serve as either substrates or inhibitors of malarial ADA and are not utilised by mammalian ADA.

Adenosine Deaminase Inhibitors↗
PubMed1985

Purine metabolic enzymes in lymphocytes. IV. Effects of enzyme inhibitors and enzyme substrates on the blastogenic responses of human lymphocytes.

Coformycin, which is an inhibitor of adenosine deaminase, significantly inhibited in vitro blastogenic responses of human lymphocytes to both phytohaemagglutinin (PHA) and pokeweed mitogen (PWM), whereas blastogenic responses to bacterial lipopolysaccharide (LPS) were rather enhanced by the addition of coformycin. Blastogenic responses of lymphocytes to PHA and PWM were markedly suppressed by the addition of adenosine, which is a substrate of adenosine deaminase. Allopurinol, which is an inhibitor of xanthine oxidase, inhibited blastogenic responses of human lymphocytes to PHA, PWM, and bacterial LPS. Inosine (a substrate of purine nucleoside phosphorylase) and hypoxanthine (a substrate of xanthine oxidase) showed no or only a small effect on blastogenic responses of human lymphocytes. These results suggest that adenosine deaminase activity is associated with the T-cell response but not with the B-cell response and that the impaired T-cell response in adenosine deaminase deficiency is the result of intracellular retention of adenosine in T cells. The results also suggest that purine nucleoside phosphorylase or xanthine oxidase activity is associated with both T- and B-cell responses.

Adenosine↗
PubMedDate not supplied

Inhibition of the zymosan-induced chemiluminescence of human phagocytes by adenosine, polyadenylic acid and agents influencing adenosine metabolism.

The zymosan-induced chemiluminescence of human phagocytes (neutrophil granulocytes) could be inhibited by adenosine, polyadenylic acid, dipyridamole (an agent blocking the cellular adenosine uptake) and coformycin (a highly potent inhibitor of enzyme adenosine deaminase). It is suggested that in estimating the antianginal and antithrombotic action of dipyridamole, its action on the phagocytes should be taken into account. The same consideration would be true for 2'-deoxycoformycin (a coformycin analogue) used as a potent anticancer and immunosuppressive drug.

Adenosine↗
PubMed1985

2-Chloroadenosine: a selective lethal effect to mouse macrophages and its mechanism.

In our studies on the effects of purine compounds on immune responses in vitro, we found that 2-chloroadenosine (2-Cl Ado) exhibited a potent lethal effect on a viability of mouse adherent cells derived from the peritoneal cavity. The lethal effect was specific for adherent peritoneal cells (PC) (macrophages) and was prevented by exogenous addition of adenosine (Ado) or coformycin, a potent inhibitor of adenosine deaminase. A rapid decrease of intracellular ATP content (26% of control) in adherent PC was observed soon after 1 hr exposure to 2-Cl Ado (0.1 mM), and this decrease of ATP was comparable with that of monoiodoacetate (MIA, 0.1 mM)- or NaN3 (5 mM)-treated adherent PC. The ATP decrease by 2-Cl Ado was restored to 88 or 90% of control value by 1 hr addition of Ado or coformycin, respectively. Polymorphonuclear cells and lymphocytes to which 2-Cl Ado did not exhibit the lethal effect did not cause a significant ATP decrease of the cells. Therefore, the data suggested that the reason for the lethal effect on adherent PC treated with 2-Cl Ado could be attributed to a rapid decrease of ATP content at an early time. We assume that 2-Cl Ado competes with intracellular Ado in macrophages and then causes the adenosine starvation resulting in the ATP decrease.

2-Chloroadenosine↗
PubMedDate not supplied

Distribution and inhibition of adenosine deaminase in tissues of man, rat, and mouse.

Adenosine deaminase activity, with arabinosyladenine (ara-A) used as the substrate, was measured in human leukemic cells and major tissues of man, rat, and mouse. The enzyme is stable in frozen tissues for at least 1 week, in frozen homogenate for several months, or after overnight dialysis at 4 degrees C in 0.25 M sucrose. The adenosine deaminase of chronic myelogenous leukemic (CML) cells has a Km of 0.6 mM and a Vmax of 183 mumols x 10(-2)/g of cells/minute. All species have highest activities in spleen and intestine. Kidney activity is highest in rat and lowest in man. The human brain has higher activity than the mouse or rat brain, and activity is higher in mouse liver than in human or rat liver. Activity in lung tissues is moderate and similar in all three species. The mean activity in CML cells is 195 mumols x 10(-2)/g of cells/minute, and the activity is higher in patients with CML in blast cell crisis. Acute myelogenous and lymphocytic leukemia cells also have very high activities. Drug concentrations required for inhibiting 50% of CML enzyme activity are 10 nM or erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), 0.6 nM of coformycin, and 0.3 nM of 2'-deoxycoformycin. Preincubation of the enzyme with coformycin, but not with EHNA, enhances the inhibition. In view of the high adenosine deaminase activities in tissues and the enhancement of the therapeutic effect of ara-A by inhibitors in mouse tumor systems, clinical trials of these combinations are warranted. They may be particularly beneficial to patients with CML in blast cell crisis.

Adenine↗
PubMed1980

Elucidation of aberrant purine metabolism: application to hypoxanthine-guanine phosphoribosylstransferase- and adenosine kinase-deficient mutants, and IMP dehydrogenase- and adenosine deaminase-inhibited human lymphoblasts.

We propose that the ratio of [14C]formate-labelled purine nucleosides and bases (both intra and extracellular) to nucleic acid purines provides, in exponentially growing cultures, a sensitive index for comparative studies of purine metabolism. This ratio was 4-fold greater for an HGPRT- mutant than for the parental HGPRT+ human lymphoblast line. The major components of the labelled nucleoside and base fraction were hypoxanthine and inosine. By blocking adenosine deaminase activity with coformycin we found that approx. 90% of inosine was formed directly from IMP rather than the route IMP leads to AMP leads to adenosine leads to inosine. The ratio of labelled base + nucleosides to nucleic acids was essentially unchagned for an AK- lymphoblast line and 2-fold greater than control for an HGPRT(-)-KAK- line, demonstrating that a deficiency of adenosine kinase alone has little effect on the accumulation of purine nucleosides and bases. Although adenosine was a minor component of the nucleoside and base fraction, the adenosine fraction increased from 3 to 13% with the addition of coformycin to the HGPRT(-)-AK- line. In the parental and HGPRT- lines, adenosine was shown to be primarily phosphorylated rather than deaminated at concentrations less than 5 microM. Inhibition of IMP dehydrogenase activity by mycophenolic acid caused a 12- and 3-fold increase in the rate of production of labelled base and nucleoside in the parent and HGPRT- cells respectively. These results suggest that a mutationally induced partial deficiency in the activities converting IMP to guanine nucleotides may result in an increased catabolism of IMP.

Adenosine Deaminase Inhibitors↗
PubMed1988

Cytosolic purine 5'-nucleotidases of rat liver and human red blood cells: regulatory properties and role in AMP dephosphorylation.

Of the various species of cellular 5'-nucleotidases, membranous, lysosomal and cytosolic, only the latter are likely to play a role in the physiologic dephosphorylation of the 5'-nucleoside monophosphates present in the cytoplasm. The necessity to preserve cellular ATP renders a strict control of the dephosphorylation as well as of the deamination of AMP mandatory, because both nucleotides are maintained in equilibrium by adenylate kinase. Our studies of cytosolic purine 5'-nucleotidases purified from rat liver and from human erythrocytes, reviewed in this presentation, have shown that both display complex kinetic properties. Both enzymes have markedly higher affinities for IMP and for GMP than for AMP. In addition, they are stimulated by nucleoside triphosphates, among them ATP and GTP, and inhibited by Pi. The erythrocytic purine 5'-nucleotidase is also stimulated by glycerate 2,3-bisphosphate. It could thus be expected that under conditions of ATP and GTP breakdown, particularly when accompanied by an increase in Pi, the dephosphorylation of AMP would be curtailed. To verify this hypothesis, experiments were performed with isolated rat hepatocytes and with human red blood cells. The rate of dephosphorylation of AMP was measured by following time-wise the production of adenosine in the presence of coformycin (or deoxycoformycin) and 5-iodotubercidin. The coformycins inhibit the deamination of adenosine into inosine by adenosine deaminase, and 5-iodotubercidin inhibits the recycling of adenosine into AMP by adenosine kinase. Upon induction of ATP catabolism by the addition of fructose to isolated rat hepatocytes, the dephosphorylation of AMP was nearly completely suppressed. In accordance with these results, the activity of the rat liver cytosolic 5'-nucleotidase, assayed in the presence of concentrations of substrate and effectors mimicking those measured in intact cells following the addition of fructose, was decreased as compared to control conditions. In hepatocytes in which ATP catabolism was induced by suppression of oxygen, the rate of dephosphorylation of AMP increased about 3-fold. However, in contradiction with these data, the activity of the cytosolic 5'-nucleotidase, measured under conditions mimicking anoxia, decreased markedly. In human erythrocytes, dephosphorylation of AMP did not occur under physiologic conditions, but proceeded when ATP catabolism was induced by glucose lack or by alkalinization. The rate of dephosphorylation of AMP was 3-fold higher during glucose deprivation than under alkaline conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Monophosphate↗
PubMed1993

Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) is a ubiquitous enzyme in eukaryotes, which may play a role in ATP catabolism during myocardial ischaemia. We report isolation of AMP deaminase from rabbit myocardium with a 19% recovery and a 650-fold enrichment, using a newly devised protocol involving sequential cation-exchange, gel-permeation and affinity chromatographies. The cardiac AMP deaminase preparation described was electrophoretically and chromatographically homogeneous and contained one unique N-terminal residue (leucine). The isolated enzyme was sensitive to various cations (K+, Mg2+, Ca2+). The pH optimum of purified cardiac AMP deaminase was 6.8, its pI was 6.5, and it displayed substrate-specificity toward 5'-AMP. The subunit molecular mass of rabbit heart AMP deaminase on SDS/PAGE (81 kDa) and the holoenzyme molecular mass as estimated by non-denaturing size-exclusion h.p.l.c. (330 kDa) indicated that the native enzyme was a tetramer. Cardiac AMP deaminase displayed a sigmoidal substrate-saturation curve in the presence of 100 mM KCl. Apparent Michaelis constants were a Km of 5.8 mM AMP and a Vmax. of 11.1 mumol/min per mg of protein. ATP and ADP were positive allosteric effectors of cardiac AMP deaminase: the apparent Km was decreased to 1.7 mM by 1.0 mM ATP. The enzyme was inhibited by GTP, coformycin, coformycin 5'-phosphate, palmitoyl-CoA, inorganic phosphate compounds, and the metal chelator o-phenanthroline. No inhibition either by product nucleotide (IMP) or by nicotinamide nucleotides was detected when these agents were examined at concentrations up to 2.5 mM. We conclude that this enzyme preparation offers a means by which the kinetic mechanism and regulation of mammalian cardiac AMP deaminase may be directly investigated.

AMP Deaminase↗
PubMed1990

Structural and conformational analysis of pentostatin (2'-deoxycoformycin), a potent inhibitor of adenosine deaminase.

X-ray, NMR and molecular mechanics studies on pentostatin (C11H16N4O4), a potent inhibitor of the enzyme adenosine deaminase, have been carried out to study the structure and conformation. The crystals belong to the monoclinic space group P21 with the cell dimensions of a = 4.960(1), b = 10.746(3), c = 11.279(4)A, beta = 101.18(2) degrees and Z = 2. The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.047 for 997 reflections. The trihydrodiazepine ring is nonplanar and adopts a distorted sofa conformation with C(7) deviated from the mean plane by 0.66A. The deoxyribose ring adopts a C3'-endo conformation, different from coformycin where the sugar has a C2'-endo conformation. The observed glycosidic torsion angle (chi = -119.5 degrees) is in the anti range. The conformation about the C(4')-C(5') bond is gauche+. The conformation of the molecule is compared with that of coformycin and 2-azacoformycin. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. The conformation of deoxyribose in solution is approximately 70% S and 30% N. Molecular mechanics studies were performed to obtain the energy minimized conformation, which is compared with X-ray and NMR results.

Adenosine Deaminase Inhibitors↗
PubMedDate not supplied

Properties of 5'-AMP deaminase and its inhibitors with the aid of a continuous fluorimetric assay with formycin-5'-phosphate as substrate.

A new continuous fluorimetric assay for AMP deaminase activity is described. The method makes use of a fluorescent analog of 5'-AMP, formycin-5'-phosphate (5'-FMP), which undergoes deamination to formycin B-5'-phosphate, not fluorescent at neutral pH. The pH-dependence for deamination of 5'-FMP is similar to that for 5'-AMP, but shifted about 0.2 units to more acidic pH. Deamination of 5'-FMP may also be followed spectrophotometrically at 306 nm, permitting better assays of crude extracts. Some kinetic results obtained by means of the new method for AMP deaminase from chick and rabbit skeletal muscle are presented. In particular it was found that the natural product of deamination, 5'-IMP exhibited allosteric inhibition of the chick enzyme with Ki values 1.6 mM, 1.2 mM and 1.0 mM at pH 5.8, 6.5 and 7.3, respectively. Activation by diadenosine tetraphosphate, Ap4A, reported for mouse muscle AMP deaminase, has not been noted for the chick enzyme. Inhibition by the transition state analogs, coformycin and 2'-deoxycoformycin, was observed for both rabbit and chick deaminases with Ki values approximately 1 microM and approximately 1.6 microM respectively. Kinetic data for coformycin-5'-phosphate show it to be a tight-binding inhibitor with Ki less than 0.6 x 10(-9) M as compared to 1 x 10(-9) M for 2'-deoxycoformycin-5'-phosphate.

AMP Deaminase↗
PubMed2002

Ring-expanded ("Fat") nucleosides as broad-spectrum anticancer and antiviral agents.

Ring-expanded (ldauo;fat") nucleosides (RENs) described in this review are analogues of purine nucleosides containing a 5:7-fused imidazodiazepine or imidazotriazepine ring system. They are both of natural and synthetic origin, and are of chemical, biochemical, biophysical, as well as medicinal interest. The important natural RENs include coformycin, pentostatin, azepinomycin, adechlorin, and adecypenol. A majority of them are synergistic antitumor and/or antiviral antibiotics which potentiate the effects of other antitumor or antiviral compounds through inhibition of key enzymes such as adenosine deaminase or guanase which would otherwise metabolically degrade the active compounds into therapeutically less potent or totally inactive counterparts. However, despite the fact that some of the natural RENs such as coformycins are the strongest known enzyme inhibitors, they have not been proven as effective clinically as anticipated because of the extremely high toxicity associated with their use. Nevertheless, pentostatin (2'-deoxycoformycin) is a conspicuous exception as it is gaining wide attention in recent years as a clinically effective therapeutic agent against leukemias and lymphomas. Many of the recently reported synthetic RENs, by contrast, possess biological activities of their own, in particular against a wide spectrum of cancers and viruses with little toxicity to the host cells, and thus hold considerable promise as chemotherapeutic agents. The promising preliminary in vitro data concerning the effects of RENs on human cancers, in particular prostate and breast cancer cells, support their further pursuit in animal and clinical studies. RENs also carry promise against many viral infections belonging to the families of hepatitis, herpes, and respiratory infections, most notable being the hepatitis B (HBV), hepatitis C (HCV), and the West Nile (WNV) viruses.

Antineoplastic Agents↗
PubMed1978

Cytotoxic and metabolic effects of adenosine and adenine on human lymphoblasts.

The metabolic and growth inhibitory effects of adenosine toward the human lymphoblast line WI-L2 were potentiated by the adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and coformycin. EHNA, 5 micron, or coformycin, 3.5 micron, at concentrations that inhibited adenosine deaminase activity more than 90% had little effect on cell growth or the metabolic parameters studied. Adenosine, 50 micron, plus EHNA, 5 micron, arrested cell growth in both parent and adenosine kinase-deficient lymphoblasts, implicating the nucleoside as the mediator of the cytostatic effect. Adenosine, 50 micron, in combination with the adenosine deaminase inhibitors reduced 14CO2 generation from [1-14C]glucose by 38%, depleted 5-phosphoribosyl-1-pyrophosphate by more than 90%, and reduced pyrimidine ribonucleotide concentrations. Uridine, 10 or 100 micron, reversed adenosine plus EHNA growth inhibition in WI-L2 but not in adenosine kinase mutants. Adenine, 500 micron, which may be converted to the same intracellular nucleotides as adenosine, reduced the growth rate by 50% in both parent and adenine phosphoribosyltransferase-deficient lymphoblasts. Although adenine also depleted cells of 5-phosphoribosyl-1-pyrophosphate and reduced pyrimidine ribonucleotide by 50%, the mechanisms of adenine and adenosine toxicity differ. In contrast to the ability of uridine to reverse adenosine cytostasis, growth inhibition by adenine was not reversed by uridine, indicating that pyrimidine ribonucleotide depletion is not the primary mechanisms of adenine toxicity.

Adenine↗
PubMed1984

Adenosine-resistant Chinese hamster fibroblast variants with hyperactive adenosine-deaminase: an analysis of the protection against exogenous adenosine afforded by increased activity of the deamination pathway.

The activity of purine salvage and interconversion enzymes was examined in two sublines of Chinese hamster cells--RA11 and RA41--isolated on the basis of their resistance to adenosine concentrations toxic to wild-type CCL39 cells. Adenosine deaminase (ADA) activity was found to be two times higher in RA11 and three times higher in RA41 than in CCL39. Inhibition of ADA activity by coformycin reduced the level of adenosine resistance but did not restore wild-type sensitivity, indicating that a second defect contributes to the adenosine-resistant phenotype of these variants; evidence was indeed obtained for the presence in both lines of additional alterations protecting them against the lethal depletion of phosphoribosylpyrophosphate (Ishii and Green, 1973) imposed by adenosine to wild-type cells. To gain better insight into the influence of ADA hyperactivity on adenosine resistance, a procedure was developed for the specific isolation of variants with increased levels of ADA activity. Cell lines with 3-5 times and then 100-500 times the wild-type ADA activity were stepwise recovered. These investigations confirmed that amplification of ADA can efficiently contribute in protecting cells against high concentrations of exogenous adenosine. The variants isolated by this procedure again manifested, in addition to amplification of ADA activity, another alteration decreasing their sensitivity to adenosine. A possible mechanism accounting for the frequent isolation of variants that coexpress ADA hyperactivity and a second defect contributing protection against adenosine toxicity are considered.

Adenosine↗
PubMed1988

Adenosine induction of rapid catabolism of adenine ribonucleotides and independent elevation of the ATP content in quiescent mouse fibroblasts.

The influence of adenosine on the ribonucleotide metabolism in quiescent BALB/c 3T3 cells was studied. The cellular adenine ribonucleotides were labelled by pretreating the cells with [2-3H]-adenine. After addition of adenosine to the cell cultures, the amount and radioactivity of the cellular purine ribonucleotides and the radioactivity of the purine compounds in the medium were determined. It appeared that adenosine gave rise both to rapid catabolism of adenine ribonucleotides with inosine 5'-monophosphate (IMP) as an intermediate and to expansion of the cellular adenosine 5'-triphosphate (ATP) pool. The maximal rates and the apparent activation constants for the two processes have been determined. Experiments with varying concentrations of coformycin (an inhibitor of adenosine 5'-monophosphate [AMP] deaminase and adenosine deaminase) and of 5'-amino-5'-deoxyadenosine (an inhibitor of adenosine kinase), respectively, showed that each compound may almost completely inhibit the adenosine-induced catabolism. This effect can be obtained under conditions where there was little or no effect by the two inhibitors on the rate of expansion of the cellular ATP pool. These results may best be explained by assuming that the process of expansion of the ATP pool is independent of the induced catabolism of adenine ribonucleotides, even though both processes seem to depend on the phosphorylation of adenosine to AMP. The total increase in the pool size of ATP and of guanosine 5'-triphosphate (GTP), both caused by adenosine, seems not to have regulatory effect on adenine ribonucleotide catabolism.

Adenosine↗
PubMedDate not supplied

Adenine nucleotide synthesis in human erythrocytes depends on the mode of supplementation of cell suspension with adenosine.

In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1-10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h. The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant. In the presence of 25-50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level. If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate. After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease. In the presence of coformycin, erythrocytes utilised adenosine at a rate of 0.2-0.3 mmol/l cells h. Their adenylate pool increased at a rate of 0.10-0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly. The energy charge of cells decreased significantly from 0.86 +/- 0.05 (control) to 0.82 +/- 0.06. Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02-5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02-0.35 mmol/l cells h. The energy charge of these erythrocytes increased significantly up to 0.91 +/- 0.03. After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2-3 h.

Adenine Nucleotides↗
PubMed1983

An enzymatic method for inosine 5'-monophosphate in the femtomole range.

A method for measuring inosine 5'-monophosphate (IMP) by enzymatic generation of NADPH is described. Procedures are given for direct fluorometric assay in the nanomole range and indirect measurement with amplification by enzymatic cycling in the pico- and femtomole ranges. The most sensitive procedure represents a nearly 50,000-fold increase in sensitivity over enzymatic methods now available. Specificity of the assay was greatly enhanced by the use of the antibiotic coformycin, a potent inhibitor of adenosine deaminase (EC 3.5.4.4). This enzyme was found to be a major contaminant of one of the necessary enzymes, phosphoglucomutase (EC 2.7.5.1). The use of the method is illustrated by measurements of IMP in single stimulated and control rat muscle fibers.

Adenosine Deaminase Inhibitors↗