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This study was aimed to evaluate the effects of intoxication of common carp with nitrite on some properties of Adenosine 5´-monophosphate (AMP) deaminase in the skeletal muscle. LC50 value was determined to be 512 mg/L. After estimating the LC50, fish were exposed to 153 mg/L (30% LC50) and sampling was done at 0, 24, 48, 72, and 96 h after exposure. Cytosolic AMP deaminase was purified from the skeletal muscle of the control and experimental groups. AMP-deaminase showed Hill kinetics with an S0.5 value for AMP of 2.52±0.031 and 1.45±0.07 mM, a Hill coefficient (nH) of 1.35±0.051 and 1.79±0.60, and a maximal velocity (Vmax) of 133.39±28 and 98.9±38 IU/mg protein for the control and experimental groups, respectively after 96 h. The optimum pH was reduced during nitrite intoxication. The maximal activation of the enzyme was at 45ºC. Also, activation energy declined in the experimental groups. Fenton reaction showed that the enzyme was tolerant to free radicals. According to the results, AMP deaminase activity in the common carp’s muscles increased following nitrite intoxication. Also, acidic condition increased AMP deaminase activity that could be an adaptive response to hypoxic condition caused by nitrite intoxication.

Background Protein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. Methods To determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. Results Overexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. Conclusions Altogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.

Shrimp muscle contains taste component compounds, such as glutamic acid and inosinic acid (IMP). When both are present, taste intensity increases by fourfold to eightfold compared to that when only glutamic acid is present. Inosinic acid is generated via adenosine triphosphate degradation in two metabolic pathways in which adenosine monophosphate (AMP) is generated and then degraded to either IMP or adenosine (AdR). We investigated post-mortem AMP degradation pathways in the shrimp Penaeus japonicus by measuring the activity of enzymes extracted from shrimp muscle and by isolating bacteria from shrimp muscle and examining their role in AMP degradation. The enzyme extract degraded AMP to IMP through high AMP deaminase activity, and in addition, we identified Shewanella sp. and Exiguobacterium sp. as mediating AMP degradation to AdR. Therefore, preventing an increase in bacterial numbers during storage is important for preventing AMP from degrading to AdR.

The mechanisms for cell toxicity with adenosine deaminase inhibition by 2'-deoxycoformycin (dCF) in non replicating lymphoid cells include S-adenosylhomocysteine (SAH) hydrolase inactivation and reduction of cellular ATP content. These postulates were explored in a patient with T-CLL receiving dCF with a resultant fall in peripheral blood lymphocytes from 740 X 10(9)/1 to 90 X 10(9)/1 over 15 d. In red cells there was complete inhibition of adenosine deaminase and SAH hydrolase activities, progressive deoxyadenosine triphosphate (dATP) accumulation and ATP depletion but no significant alteration in adenosine monophosphate (AMP) deaminase activity or distribution in purine intermediates from radioactive adenosine. In T-CLL lymphocytes, there was incomplete lymphoid SAH hydrolase inactivation, reduced AMP deaminase activity and progressive dATP accumulation. The limited decrease in lymphocyte ATP content was related more to dCF administration than dATP accumulation, nor accompanied by significant changes in the distribution of purine intermediates from adenosine. These findings suggest that ATP depletion with dCF therapy does not reflect AMP deaminase activity modulation nor is of critical importance for cell toxicity. The exact role for elevated cellular dATP content and SAH hydrolase inactivation in this toxicity remains to be established.

AMP-deaminase was purified to electrophoretic homogeneity from white skeletal muscle of a teleost fish, the common carp, Cyprinus carpio. The purified enzyme was highly stable and showed non-Michaelis-Menten kinetics with a S(0.5) value for AMP of 2.52+/-0.16 mM (SEM) and a Hill coefficient of 1.19+/-0.11. Specific activity of the purified enzyme was 1000-1200 U/mg protein. The pH optimum was 6.3 and the enzyme was activated by ADP and ATP, but inhibited by phosphate and fluoride. Low concentrations of NaCl and KCl (100-150 mM) activated, whereas higher concentrations were inhibitory. Free radicals inactivated the enzyme, decreasing V(max) by one-half but not affecting S(0.5) or Hill coefficient. Possible regulatory mechanisms of AMP-deaminase activity in fish muscle are discussed.

The characteristics of flounder (Paralichthys patagonicus) were evaluated during a 12-day period of storage in ice by monitoring the biochemical, microbiological and sensory parameters. The adenosine-5-triphosphate and adenosine-5-diphosphate were not detected, and the initial content of adenosine-5-monophosphate (AMP) rapidly decreased to zero. Inosine-5monophosphate (IMP) but not adenosine (Ado) was present in muscle extracts. The AMP deaminase activity was found in the muscle. Conversely, the Ado deaminase activity was not detected. A 55% decrease in IMP concentration occurred during the first 2 days of storage. The inosine content was barely detectable (0.1 mmol/g), probably because of the high nucleoside phosphorylase activity in the muscle. Hypoxanthine significantly (P 0.01) increased up to the seventh day and then remained approximately constant. The muscle pH fluctuated between 6.6 and 7.0. The iced flounder was microbiologically acceptable for up to 7 days with aerobic plate counts reaching 10 7 cfu/g. The sensory score, determined by the Quality Index Method, linearly increased (r 2 = 0.96) with the keeping time. All the parameters, including the K-value, indicated that the shelf life of iced flounder was 7 days.

The usefulness of in vitro AMP deaminase inhibition was examined as a potential molecular method in risk assessment of xenobiotics. The enzyme participates in the principal purine nucleotide interconversion and degradation pathways, and its absence caused perturbations in the cellular ATP pool. The compounds selected were synthetic musks with a known negative environmental impact and the toxicologically unknown ionic liquids and N -glucopyranosyl ammonium bromides, which have recently attracted much interest from the chemical and related industries. All the compounds tested demonstrated a dose-dependent inhibition of AMP deaminase activity. IC 50 ranged from 0.3 μM for polycyclic musks to 500 μM for N -glucopyranosyl trimethylammonium bromide. Analysis of Dixon plots showed the inhibition type for all the compounds to be noncompetitive. The results support the choice of such an assay for the prospective risk assessment of these compounds.

Abnormal elevations in ammonia have been implicated in the pathogenesis of Alzheimer’s disease. However, the biochemical mechanism(s) leading to increased ammonia in Alzheimer’s disease have not yet been identified. A potential source of increased ammonia production is adenosine monophosphate (AMP) deaminase, an important enzyme in the regulation of the purine nucleotide cycle and adenylate energy charge. AMP deaminase activity is expressed in human brain and converts AMP to inosine monophosphate with the release of ammonia. We have investigated AMP deaminase activity in postmortem brain tissue from Alzheimer’s disease subjects and age-matched controls. Compared to control brain, Alzheimer’s disease brain AMP deaminase activity is 1.6- to 2.4-fold greater in the regions examined—the cerebellum, occipital cortex, and temporal cortex. Similar increases in AMP deaminase protein and mRNA levels are observed in Alzheimer’s disease brain. These results suggest that increased AMP deaminase activity may augment ammonia levels in the brain in Alzheimer’s disease.

A new, sensitive assay for the quantitative determination of AMP deaminase activity in human skeletal muscle is presented. The method is based on the determination of the direct product of the AMP deaminase reaction, the formed IMP, by high performance liquid chromatography (HPLC). In order to evaluate the relationship between AMP deaminase activity on the one hand and the contractile and metabolic characteristics of the muscle and the physical performance on the other, muscle biopsies were taken from 20 male subjects. The subjects also performed a 30 s sprint test on a cycle ergometer. The inter-individual variation in AMP deaminase activity was large, ranging from 5.4 to 27.4 microkat g-1 dry muscle. AMP deaminase was positively correlated with phosphofructokinase (PFK), the marker of the glycolytic capacity of the muscle, but there was no correlation with enzymes of oxidative metabolism, such as 3-hydroxyacyl-CoA dehydrogenase and citrate synthase, or with the activity of myokinase and lactate dehydrogenase. There was no significant correlation between AMP deaminase activity and the proportion of the different muscle fibre types. A weak positive correlation was found between the sprint performance and the AMP deaminase activity. In conclusion, the HPLC assay was found to be a fast, sensitive and reliable method for the quantitative determination of AMP deaminase activity in muscle. A direct relationship between AMP deaminase activity on the one hand and glycolytic capacity and sprint performance on the other was found. However, no relationship to oxidative capacity or contractile properties was found.

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.

The interaction of pig heart AMP deaminase with different chemical species of phosphatidylcholine and with natural plasma membranes has been investigated. Phospholipids added to the system either as natural biological membranes (plasma membrane vesicles) or in the form of liposomes containing unsaturated phosphatidylcholine considerably enhanced AMP deaminase activity. The secondary structure of pig heart AMP deaminase in the absence and in the presence of dioleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine liposomes was investigated by Fourier-transform infrared spectroscopy. Quantitative analysis of the amide I band showed that the enzyme contains 45% beta-sheets, 28% alpha-helix, 16% turns and 11% non-ordered structure. In the presence of dioleoyl phosphatidylcholine liposomes, the beta/alpha content ratio decreased; this decrease was dependent on the amount of lipid added. This phenomenon was not observed in the case of dipalmitoyl phosphatidylcholine liposomes. These data suggest a possible role for membrane phospholipids in the regulation of AMP deaminase activity.

In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p

Cardiac matrix metalloproteinases (MMPs) stimulated by the sympathomimetic action of angiotensin II (AII) exacerbate chamber diastolic stiffening in models of subacute heart failure. Here we tested the hypothesis that MMP inhibition prevents such stiffening by favorably modulating high- energy phosphate (HEP) stores more than by effects on matrix remodeling. Dogs were administered AII i. v. for 1 week with tachypacing superimposed in the last two days (AII + P; n = 8). A second group (n = 9) underwent the same AII + P protocol but was preceded by oral treatment with an MMP inhibitor PD166793 [(S)-2-(4-bromo-biphenyl-4-sulfonylamino-3-methylbutyricacid] 1 week before and during the AII + P period. Pressure-volume analysis was performed in conscious animals, and myocardial tissue was subjected to in vitro and in situ zymography, collagen content, and HEP analysis (high-performance liquid chromatography). As reported previously, AII + P activated MMP9 and MMP2 and specifically exacerbated diastolic stiffening (+ 130% in chamber stiffness). PD166793 cotreatment prevented these changes, although myocardial collagen content, subtype, and cross-linking were unaltered. AII + P also reduced ATP, free energy of ATP hydrolysis (Delta G(ATP)), and phosphocreatine while increasing free [ADP], AMP catabolites (nucleoside-total purines), and lactate. PD166793 reversed most of these changes, in part due to its inhibition of AMP deaminase. MMP activation may influence cardiac diastolic function by mechanisms beyond modulation of extracellular matrix. Interaction between MMP activation and HEP metabolism may play an important role in mediating diastolic dysfunction. Furthermore, these data highlight a potential major role for increased AMP deaminase activity in diastolic dysfunction.

Possession of the nonsense mutation in AMPD 1 C34T gene has been linked to improved survival in patients with heart failure, possibly by promoting the formation of adenosine. This mutation is known to decrease the activity of AMP-deaminase in skeletal muscle. We have found that the AMPD1 mutation decreases the activity of AMP-deaminase in the heart without changing the activity of any other enzymes of adenine nucleotide metabolism. Protective mechanism of this mutation may be thus induced by local cardiac metabolic changes.

Dietary supplementation of the creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases in vitro skeletal muscle AMP deaminase (AMP-D) activity in rats. Downregulation of AMP-D activity was progressive and greater in fast-twitch muscles (70-80%) than in the slow-twitch soleus muscle (approximately 50%). The loss in AMP-D activity had little effect on inosine 5'-monophosphate accumulation in mixed-fiber muscle with intense tetanic contractions. In contrast, inosine 5'-monophosphate formation was evident earlier in fast-twitch red and white fiber sections of creatine-depleted animals during intense twitch contractions, indicating that fast-twitch muscle of beta-GPA-treated rats buffers decreases in the ATP/ADPfree ratio via deamination, even though AMP-D activity is less. Isoforms of skeletal muscle AMP-D mRNAs in mixed-fiber muscle were not altered by feeding beta-GPA for up to 9 wk. Creatine depletion did not alter total immunoreactivity; however, a redistribution of AMP-D immunoreactivity from primarily an approximately 80-kDa form toward lower apparent molecular mass species (approximately 60 and approximately 56 kDa) was observed. Posttranslational changes in AMP-D appear related to changes in activity.

In 1978 adenosine monophosphate (AMP) deaminase deficiency in skeletal muscle (“myoadenylate deaminase deficiency”) was described as a new entity by Fishbein et al. Their publication, “Myoadenylate deaminase deficiency: a new disease of muscle,” reported five patients with muscular weakness or cramping after exercise. Muscle biopsies of all five patients showed less than 5% of normal AMP deaminase activity, levels of other enzymes were normal. Routine histology of skeletal muscle was also normal. The symptoms of these patients were attributed to the lack of AMP deaminase activity.

A novel method for measuring AMP-deaminase activity in human erythrocytes is presented, based on the determination of the reaction product, IMP, using high performance liquid chromatography. IMP formation was found to be proportional both to the incubation time and the amount of haemolysate over a wide range. The minimal detectable AMP-deaminase activity was more than 1000 times lower than the mean activity found in healthy controls (1083 nmol/h/mg Hb). No marked difference of activity was found in the patients with the following inherited purine disorders: familial juvenile gouty nephropathy and deficiencies of adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase or adenine phosphoribosyltransferase. The activity in the erythrocytes of patients with chronic renal failure was also similar to controls. The existence of subjects with low erythrocyte AMP-deaminase activity in the population has been confirmed.

The regulation of platelet AMP deaminase activity by ATP, GTP and phosphate was studied in human platelets in situ, and in vitro after partial purification. In intact platelets, a similar 50% decrease in cytosolic ATP was induced by either glucose starvation or treatment with H2O2. During starvation, AMP deaminase was in the inhibited state, as ATP consumption was mostly balanced by the accumulation of AMP. During H2O2 treatment, however, the enzyme was in the stimulated state, as the AMP formed was almost completely deaminated to IMP. Cytosolic GTP fell by 40-50% in both starvation and H2O2 treatment. In contrast, intracellular phosphate was 4-5-fold higher in starved than in H2O2-treated cells. These data point to phosphate as the main regulator of AMP deaminase activity in situ. This conclusion was verified by kinetic analysis of partially purified AMP deaminase. At near-physiological concentrations of MgATP, MgGTP and phosphate, the S0.5 (substrate half-saturation constant) for AMP was 0.35 mM. Half-maximal stimulation by MgATP occurred at a concn. between 2 and 3 mM. This stimulation was antagonized by the inhibitory effects of phosphate (IC50 = 2.0 mM) and MgGTP (IC50 = 0.2-0.3 mM), which acted in synergism (IC50 is the concentration causing 50% inhibition). We conclude that the difference in adenylate catabolism between starved and H2O2-treated platelets is due to the distinct phosphate concentrations. During starvation, refeeding and H2O2 treatment, the values of the adenylate charge and the phosphorylation potential were kept closely co-ordinated, which may be effected by AMP deaminase.

The physiological role of the inhibition of AMP deaminase (EC 3.5.4.6) by Pi was analyzed using permeabilized yeast cells. (a) Fructose 1,6-bisphosphatase (EC 3.1.3.11) was inhibited only a little by AMP, which was readily degraded by AMP deaminase under the in situ conditions. (b) The addition of Pi, which showed no direct effect on fructose 1,6-bisphosphatase, effectively enhanced the inhibition of the enzyme by AMP increased through the inhibition of AMP deaminase. (c) Pi activated phosphofructokinase (EC 2.7.1.11) and inhibited AMP deaminase activity. AMP deaminase reaction can act as a control system of fructose 1,6-bisphosphatase activity and gluconeogenesis/glycolysis reaction through the change in the AMP level. Pi may contribute to the stimulation of glycolysis through the inhibition of fructose 1,6-bisphosphatase by the increase in AMP in addition to the direct activation of phosphofructokinase.

A sensitive, optimized assay for serum AMP deaminase (EC 3.5.4.6) is presented, which is based on a colorimetric indophenol determination of ammonia liberated during the enzyme reaction. An average enzyme activity in serum of 2.7 U/liter (n = 36, range 0-6.2) for healthy adults was established. Both the enzyme and colorimetric reactions were optimized to give an assay sensitivity of 0.2 U/liter of serum, a 98-99% analytical recovery in the normal serum enzyme concentration range, and a CV of 7.9% in-run and 10.6% between-run. This optimization included studies on specific buffer, pH, ionic strength, and potential activators. The Km for the enzyme in serum was determinated to be 1.4 X 10(-3) mol/liter, which agrees well with the value reported for human skeletal muscle enzyme. Above-normal serum AMP deaminase activity may be present in various myopathies and could be useful in detecting carriers of the X-linked dystrophies.

IMP production in and force exerted by rat quadriceps muscle in situ during various types of exercise were examined in relation to age. During continuous isometric exercise with constant stimulation time, the amount of IMP was linearly and inversely related to the age of the animals; a higher IMP concentration was found in intermittent isometric and dynamic exercise. No relationship was found between the total AMP deaminase activity and age. Exercise influenced neither the total activity nor the activity in the soluble fraction. From the results it is concluded that: the IMP concentration is linearly related to the free intracellular ATP4-/ADP3- ratio and the free AMP2- concentration; older animals are better able to maintain a high intramuscular ATP4-/ADP3- ratio and a low AMP2- concentration; IMP is produced in particular under conditions when the muscle has to work under extreme stress. IMP possibly exerts a feed-back control on the contraction system.

Thrombin-stimulated platelet secretion is accompanied by a 30% reduction in the steady state level of cytosolic ATP, a breakdown that proceeds through ADP, AMP, IMP, and inosine to hypoxanthine. The ATP to hypoxanthine conversion could be blocked at the stage of AMP deamination by incubation of platelet-rich plasma for 6 h with 200 microM coformycin, a transition-state analog inhibitor of AMP deaminase. Abolition of AMP deaminase activity had no effect on thrombin-induced secretion from the dense granules, alpha-granules, or acid hydrolases measured in gel-filtered platelets. Coformycin treatment had no effect on thrombin-stimulated lactate production, even when oxidative phosphorylation was blocked by antimycin A, nor on the rate of thrombin-stimulated glycogenolysis. In addition, although it was clear that the adenylate energy charge was maintained by activation of AMP deaminase following thrombin treatment, the adenylate energy charge was also maintained in coformycin-treated platelets, albeit after a short lag, by stimulated ATP production and equilibration through the adenylate kinase reaction. Hydrogen peroxide brings about similar adenylate degradation which could also be inhibited by coformycin. The results indicate that AMP deamination and secretion, although temporally related, are not coupled. The role of AMP deaminase appears to be to maintain the adenylate energy charge in the absence of stimulation of ATP production or to buffer the adenylate charge before ATP production is stimulated.

1. 1. SAMP lyase and AMP deaminase were determined in parenchymal and kupffer cells of rats fed either basal or carcinogen-enriched diets. Results were calculated on a U/mg protein and U/cell basis. 2. 2. Data indicated that although deaminase increased 1 1 2 - to 2-fold in parenchymal cells on a U/mg protein and U/cell basis from rats fed carcinogen-enriched diets there was a greater increase in U/mg protein. 3. 3. In contrast, little to no increase was seen in kupffer cells. 4. 4. SAMP lyase, however, depicted a smaller increase in parenchymal cells of carcinogen-enriched diet fed rats, but a 4- to 5-fold elevation in kupffer cells regardless of whether the data were expressed in U/mg protein or U/cell. 5. 5. These data indicate that increased activity of AMP deaminase may be a result of resistance to degradation in parenchymal cells, whereas SAMP lyase elevations in kupffer cells may reflect an increase in enzyme concentration.

Phosphatidate bilayers composed of dilauroylphosphatidate, dimyristoylphosphatidate, dipalmitoylphosphatidate and dioleoylphosphatidate were prepared. Their interaction with AMP deaminase isolated from pig heart was investigated. Dioleoylphosphatidate bilayers were found to exert non-competitive inhibition on the AMP deaminase with a Ki of 15 x 10(-6) M. This inhibition is three orders of magnitude stronger than that exerted by orthophosphate. The phosphatidate species containing saturated fatty acids were either non-inhibitory or inhibited enzyme activity rather poorly. However, alkalinization of the medium from pH 6.5 to pH 7.9 led to the inhibition of pig heart AMP deaminase by dilauroylphosphatidate bilayers. This was accompanied by the fluidization of the saturated phosphatidate species, i.e. the lowering of their phase transition temperature in alkaline pH, as measured by light-scattering and fluorescence scans. The possible significance of these findings for the regulation of AMP deaminase activity in vivo by natural membranes is discussed.

1. 1. The molecular correlation concept proposed that IMP dehydrogenase activity should be a sensitive target of chemotherapy. This hypothesis received support from an array of evidence. IMP dehydrogenase has the lowest activity in purine biosynthesis; it is the rate-limiting enzyme in GTP production; the enzymic activity is transformation- and progression-linked; it is elevated in all examined animal and human neoplastic cells. The activity of GMP synthetase and the concentrations of GMP and dGTP were increased in cancer cells. Whereas guanine salvage has a high potential activity, the low guanine content may well curtail actual salvage capacity. Ribonucleotide reductase activity was two orders of magnitude lower than that of IMP dehydrogenae. 2. 2. Tiazofurin, a C-nucleoside, had marked cytotoxicity on hepatoma cells in vitro and was the first drug that as a single agent profoundly inhibited the proliferation of the subcutaneously inoculated solid hepatoma 3924A in the rat. 3. 3. The impact of tiazofurin administration in hepatoma cells was revealed in a cascade of biochemical alterations involving primary, secondary and tertiary targets and markers of this drug action. The primary target was IMP dehydrogenase where the active metabolite of tiazofurin, TAD, was thought to be absorbed to the NADH site of the enzyme. As a consequence, the enzymic activity declined rapidly to about 30–40% and returned to normal range by 36 to 48 hr after injection. The secondary targets and markers are the profoundly decreased pools of guanylates (GMP, GDP, GTP). Concurrently, the concentrations of IMP and PRPP were increased 8- to 15-fold. The elevated IMP pools were attributed to the de-inhibition of the AMP deaminase activity subsequent to the decline in GTP concentration. The rise in PRPP pools was attributed to the selective inhibition of GPRT and HPRT activities by the high IMP pool which did not affect APRT activity. This interpretation is supported by the 6- to 8-fold increase in the concentrations of guanine and hypoxanthine and the lack of change in the adenine pools in the hepatomas after tiazofurin administration. The marked drop in NAD concentration which was drug dose- and time-dependent is attributed to the competition for NAD pyrophosphorylase activity by the precursors of NAD and tiazofurin monophosphate. The tertiary targets were dominated by the profound alterations in the concentrations of the dNTPs. This was characterized by a rapid and persistent drop (for 3 days) of the dGTP pool. The concentrations of dATP and dCTP also declined, but these alterations were less pronounced and the pools returned to normal after 2 days. In contrast, dTTP concentrations were continuously increased. The decline in the dNTP pool is attributed to desaturation by the decreased GDP concentration of ribonucleotide reductase. The increased dTTP concentration is attributed to the thymidine kinase activity which is highly elevated in the hepatoma. This interpretation is supported by the presence of markedly enlarged pools of thymidine and thymine in the hepatomas of tiazofurin-treated rats. 4. 4. The determinants of selectivity of tiazofurin action in hepatoma and liver entail the high concentrations of TAD observed in the hepatoma after tiazofurin injection. This is attributed to the high ratios of the activities of NAD pyrophosphorylase/phosphodiesterase in the tumors. Moreover, the hepatomas had low concentrations of NAD which decreased further after tiazofurin injection; there was no change in the liver. The IMP concentration in the hepatoma was markedly lower than that of the liver. The biochemical response to tiazofurin was much more pronounced in the hepatoma, whereas in the liver there was no change in the concentrations of guanylates and only a minor increase in IMP concentration. 5. 5. The results emphasize the differences in the biochemical phenotypes and responsiveness to tiazofurin of sensitive and non-sensitive tissues. The identification to tiazofurin of sensitve and non-sensitive tissues. The identification of targets and markers of tiazofurin suggest that for rapid screening of tiazofurin sensitivity the most meaningful indicators should be the drug-induced elevations in the concentrations of TAD and in the ratios of IMP/GTP or IMP/dGTP. Further indicators include a markedly depressed NAD concentration and a strongly elevated PRPP pool in the sensitive tissues after tiazofurin treatment.

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.

A new continuous fluorimetric assay for AMP deaminase activity is described. The m ethod makes use of a fluorescent analog of 5′-AMP, formycin-5′-phosphate (5′-FM P), which undergoes deam ination 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′-FM P 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 K, values 1.6mM , 1.2mM and 1.0mM 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 ~ 1 μM and ~ 1.6 μM respectively. Kinetic data for coformycin-5′-phosphate show it to be a tight-binding inhibitor with K, < 0.6 × 10-9 M as compared to 1 × 10-9 m for 2′-deoxycoformycin-5′-phosphate.

Lactate (LDH) and succinate (SDH) dehydrogenases activities decreased in red and white muscles of rat under acute ethanol loading indicating the inhibition of energy metabolism and stepped up lactic acid formation under stress conditions. Aspartate aminotransferase (AAT) and glutamate dehydrogenase (GDH) were found to increase. In contrast to these, the AMP deaminase activity decreased in white muscle suggestive of decreased deamination of nucleic acids. The ornithine cycle enzymes such as argininosuccinate synthetase (ArSS) and arginase indicated diminished activities showing low level of operation of urea cycle and consequent accumulation of ammonia was observed in red muscle with low production of glutamine, whereas in the case of white muscle this trend is reversed. The possible alterations of ethanol toxicity on energy requirements, transdeamination patterns, ureogenesis and glutamine production have been discussed.

1. 1. The AMP deaminase activity present in a crude extract of trout gill increased with time. 2. 2. Soybean trypsin inhibitor and α 2 -macroglobulin inhibited the AMP deaminase activation. NEM was also inhibitory at 10 −3 M. 3. 3. The activation process is followed by a decrease of activity which is inhibited by EGTA and enhanced by Mg 2+ . These two compounds were without effect on the activation process itself. 4. 4. Trypsin induces a sharp activation of AMP deaminase in a fresh gill extract but is without effect on a fully activated extract. 5. 5. Overall, the results suggest that neutral proteinases are implicated in AMP deaminase activation.

The enzymes of the purine nucleotide cycle-AMP deaminase, adenylosuccinate synthetase, and adenylosuccinate lyase-were examined as a functional unit in an in vitro system which simulates the purine nucleotide composition of sarcoplasm. Activity of each cycle enzyme in extracts of rat skeletal muscle was observed to increase as ATP/ADP, reflecting the energy state of the system, was lowered from approximately 50 to 1. The increase in AMP deaminase activity could be attributed to effects of energy state and factors such as AMP concentration, which are obligatorily coupled to energy state. The increases in synthetase and lyase activities were accounted for by increases in the concentration of IMP and adenylosuccinate, respectively. The inhibitory influence of IMP concentration on synthetase activity reported in other systems was not observed in this system; synthetase activity progressively increased as IMP concentration was raised to approximately 4 mM, and apparent saturation occurred at concentrations above 4 mM. Also, adenylosuccinate was found to be an activator of AMP deaminase. The results of this study document that the activities of the enzymes of the purine nucleotide cycle increase in parallel at low energy states, and the components of the cycle function as a coordinated unit with individual enzyme activities linked via concentrations of cycle intermediates.

A procedure is described for purifying adenylate deaminase from calf brain. The method involves salt precipitation from the soluble protein of brain followed by heat treatment, diethylaminoethyl cellulose column chromatography, and either sucrose density gradient centrifugation or gel filtration. The final purification is 180-fold. The enzyme is activated by adenosine triphosphate and by alkali metal ions. The most effective alkali metal ion is that of lithium. Ammonium and tetramethylammonium ions have no effect on activity. The activators do not alter the pH optimum of the enzyme, which is about pH 6.2. ATP protects the enzyme against heat inactivation. Some kinetic properties of the enzyme in the presence and in the absence of ATP and LiCl are presented. A plot of the reaction rate with respect to the AMP concentration in the absence of activators yields a curve with a pronounced sigmoidal shape. Addition of LiCl yields similar curves which are displaced to the left. Addition of ATP yields curves which approximate rectangular hyperbolas. The effect of both activators is thus to increase the apparent affinity of the enzyme for AMP without affecting the maximum reaction velocity. Conversely, increasing the concentration of AMP has the effect of increasing the apparent affinity of the enzyme for ATP. Addition of LiCl also increases the apparent affinity of the enzyme for ATP.

The objective in these experiments was to determine whether increases in hepatic AMP deaminase activity after ingestion or injection of the hepatocarcinogens, 3'-methyl-4-dimethylaminoazobenzene or thioacetamide, were associated with increased enzyme amount or alterations in allosteric modulation. Using crude 105,000 g supernatant fractions from liver homogenized in 0.25 M sucrose as the enzyme source, data were obtained which indicated that increased AMP deaminase activity was associated with increased enzyme amount. Data in support of this contention were: (1) activity of enzyme from livers of control or hepatocarcinogen-treated animals was modulated by alkali metal ions and by certain mono-, di- or triphosphonucleotides, but the extent and nature of these modulations were similar, irrespective of enzyme source; (2) when reaction mixtures contained ATP and alkali metal ions, plots of velocity versus substrate concentration were hyperbolic and activity of enzyme from hepatocarcinogentreated animals always exceeded that of controls; and (3) reaction of enzyme from control or hepatocarcinogen-treated animals with antibody to partially purified AMP deaminase from rat liver resulted in complete loss of enzyme activity, irrespective of source, but titration curves indicated higher antigen levels in supernatants from hepatocarcinogen-treated rats. Incubation of 105,000 g supernatants at 55° resulted in losses of AMP deaminase activity, but enzyme activity losses among supernatants from carcinogen-treated animals occurred at rates nearly 3-fold greater than losses from control supernatants. Thus, it appeared that, concomitant with induction of AMP deaminase by hepatocarcinogens, appreciable alterations in physical properties of the enzyme occurred.

A rapid method for the preparation of crystalline 5'-adenylic acid deaminase from rabbit skeletal muscle is presented. The enzyme remains bound to cellulose phosphate under conditions at which apparently no other proteins are bound; thus it was possible to develop, in essence, a one-step method for its purification. The crystalline preparation is homogeneous as indicated by its elution profile, by ultracentrifugation, and by acrylamide gel electrophoresis. The new method results in enzyme that has 5 times the specific activity of the previously reported crystalline preparation and differs in stability and kinetic properties. The enzyme is stable in dilute solution for extended periods at room temperature provided the diluting medium is of high ionic strength and contains β-mercaptoethanol. The enzyme follows normal Michaelis-Menten kinetics with respect to adenosine monophosphate concentration when assayed in the presence of KCl, and with respect to potassium concentration. Sodium and potassium, at 0.15 m, function equally efficiently as cation activators.

In the physiological range of the adenylate energy charge in liver (0.7 to 0.9) the rate of deamination of AMP catalyzed by rat liver adenylate deaminase (EC 3.5.4.6) increases sharply with decreasing energy charge. It is suggested that this response serves to protect against sharp transient decreases in energy charge: when the charge decreases the resulting removal of AMP by deamination will oppose the decrease in charge (the mole fraction of ATP plus half the mole fraction of ADP). The activity of the enzyme decreases sharply as the size of the adenine nucleotide pool decreases in and below the physiological range. This effect may be a self-limiting response to prevent excessive depletion of the pool. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on liver in vivo reported by other workers.

AMP deaminase, which catalyzes the deamination of AMP to form IMP and NH3, has been known to be widely distributed in various kinds of animal tissue, however, its presence in the muscles of some invertebrates such as mollusca has been questioned. The author has detected AMP deaminase activity in the muscle of the abalone, Haliotis discus hannai, and partially purified the enzyme by high speed centrifugation, ethanol fractionation, Li2SO4 precipitation and chromatography on DEAE cellulose. The partially purified enzyme is rapidly inactivated in the absence of ATP by dialysis and heat. ATP is considered to activate and simultaneously to stabilize this enzyme effectively. The optimum pH was found to be near pH 6.4 in citrate Tris buffer. The enzyme is specific for 5'-AMP.

1. 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. 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.

Deoxyadenosine and its nucleotides are thought to be involved in the pathogenesis of the immunodeficient state associated with inherited adenosine deaminase (ADA) deficiency (1–3) and in the lymphotoxicity that occurs during the treatment of leukemia with deoxycoformycin, an ADA inhibitor (1–3). Lymphotoxicity can be simulated in vitro by treating lymphoid cells with deoxyadenosine in the presence of an ADA inhibitor. Such a treatment of lymphoblasts results in intracellular accumulation of deoxy-ATP (dATP), inhibition of DNA synthesis, ATP depletion, leading to cell toxicity; different lymphoid cells display very different sensitivity to this deoxyadenosine cytotoxicity (4, 5).The mechanism of dATP-induced cytotoxicity is still poorly understood; one of the first hypotheses was the blockade by dATP of ribonucleotide reductase which would result in an inhibition of DNA synthesis due to the shortage of deoxynucleotide precursors (4–6). More recently it was claimed that a dATP-induced inhibition of RNA synt...

From a mutagenized population of wild type S49 cells, a clone was isolated in a single step that possessed functional and biochemical deficiencies in both AMP deaminase and thymidine kinase activities. This mutant cell line, DTB6, was selected in semi-solid medium containing 1mM thymidine and 1mM dibutyryl cyclic AMP. In comparative growth rate experiments, DTB6 cells were considerably less sensitive than parental cells to the growth inhibitory effects of thymidine. In contrast, DTB6 cells were much more sensitive to the cytotoxic effects of adenine and adenosine. The supersensitivity of DTB6 cells toward adenine could be ameliorated by the addition of hypoxanthine to the culture medium. The growth phenotype of the mutant cells could be attributed to deficiencies in two enzyme activities. First, DTB6 cells possessed a 60-70% deficiency in AMP deaminase activity, although the residual activity appeared kinetically similar to the wild type enzyme. Second, DTB6 cells possessed a virtual complete deficiency in thymidine kinase activity. Both enzyme deficiencies behaved in a recessive fashion in intraspecies hybrids. Revertants of DTB6 cells possessed wild type levels of AMP deaminase activity but remained deficient in thymidine kinase activity, while another revertant of DTB6 cells expressed 11% of the wild type thymidine kinase level but did not perceptibly change its AMP deaminase activity. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism.

3-Deazaadenine, 3-deazaadenosine, and the carbocyclic analog of 3-deazaadenosine produced similar effects on nucleotide pools of L1210 cells in culture: each caused an increase in IMP and a decrease in adenine nucleotides and had no effect on nucleotides of uracil and cytosine. Concentrations of 50-100 microM were required to produce these effects. Although 3-deazaadenosine and carbocyclic 3-deazaadenosine are known to be potent inhibitors of adenosylhomocysteine hydrolase, the effects on nucleotide pools apparently are not mediated via this inhibition because they are also produced by the base, 3-deazaadenine, and because the concentrations required are higher than those required to inhibit the hydrolase. Cells grown in the presence of 3-deazaadenine or 3-deazaadenosine contained phosphates of 3-deazaadenosine (the mono- and triphosphates were isolated); from cells grown in the presence of the carbocyclic analog of 3-deazaadenosine, the monophosphate was isolated, but evidence for the presence of the triphosphate was not obtained. A cell-free supernatant fraction from L1210 cells supplemented with ATP catalyzed the formation of monophosphates from 3-deazaadenosine or carbocyclic 3-deazaadenosine, and a cell-free supernatant fraction supplemented with 5-phosphoribosyl 1-pyrophosphate (PRPP) catalyzed the formation of 3-deaza-AMP from 3-deazaadenine. Adenosine kinase apparently was not solely responsible for the phosphorylation of the nucleosides because a cell line that lacked this enzyme converted 3-deazaadenosine to phosphates. No evidence was obtained that the effects on nucleotide pools resulted from a block of the IMP-AMP conversion, but the results could be rationalized as a consequence of increased AMP deaminase activity. This explanation is supported by two observations: (a) coformycin, an inhibitor of AMP deaminase, prevented the effects on nucleotide pools, and (b) 3-deazaadenine decreased the conversion of carbocyclic adenosine to carbocyclic ATP and increased its conversion to carbocyclic GTP. The latter conversion requires the action of AMP deaminase and the observed effects can be rationalized by a nucleoside analog-mediated increase in AMP deaminase activity. Because these effects on nucleotide pools are produced only by concentrations higher than those required to inhibit adenosylhomocysteine hydrolase, they may not contribute significantly to the biological effects of 3-deazaadenosine or carbocyclic 3-deazaadenosine.(ABSTRACT TRUNCATED AT 400 WORDS)

1. 1. The AMP deaminase isoenzymes from trout gill were activated by sodium and potassium, sodium being the most efficient. The optimal concentration for activation was 30–50 mM. 2. 2. The enzyme was sensitive to ionic strength, and imidazole was an inhibitor at concentrations higher than 25 mM. A possible regulation of gill AMP deaminase by intracellular imidazole buffers is discussed. 3. 3. AMP deaminase activity was tested in the presence of physiological concentrations of sodium and potassium. When the concentration of one of these cations was varied around its physiological concentration, the enzyme activity was relatively stable, indicating that the intracellular AMP deaminase activity would be insensitive to changes in the concentrations of monovalent cations. 4. 4. The effects of the sodium salts of different inorganic and organic anions were tested. Except chloride and gluconate, all were inhibitors of gill AMP deaminase.

Publisher Summary This chapter presents evidence for a peptidic AMP deaminase activator in the gill of trout. The AMP deaminase activity present in a trout gill extract homogenized by sonication or by running an Ultra-Turrax homogenizer increases with time. This phenomenon is related to the action of proteolytic enzymes. There is no AMP deaminase activation if a smooth homogenization procedure is used. The results indicate that the activating proteinases are located in the 20000 g pellet. When sonication is replaced by treatment with digitonine, AMP deaminase activation takes place in the 900 g supernatant incubated at 25°C. The activator is not dialyzable and is TCA-precipitable. It is inactivated by treatment with trypsin or papain. This indicates that the activator is probably of peptidic nature. The results are in favor of the presence in trout gill epithelium of an AMP deaminase activating process in which are implicated (1) a peptidic activator and (2) neutral proteinases.

The changes in ammonia (NH3) and amino acid contents in human skeletal muscle during isometric exercise (2/3 maximal voluntary contraction force) to fatigue have been investigated. Biopsies from musculus quadriceps femoris were obtained at rest, fatigue, and 1 and 4 min recovery. Muscle NH3 (n = 9) increased from 1.3 +/- 0.3 (mean +/- SE) mmol/kg dry muscle (dm) at rest to 3.6 +/- 0.6 at fatigue (P less than 0.01) and remained elevated during recovery, whereas the lactate increase after contraction decreased rapidly during recovery. Total adenine nucleotide (TAN) content decreased from 28.7 +/- 0.5 mmol/kg dm at rest to 25.1 +/- 0.6 at fatigue (P less than 0.001). Muscle glutamine did not change after contraction (P greater than 0.05), whereas glutamate decreased (P less than 0.001), and alanine increased (P less than 0.001). In vivo AMP deaminase activity (measured by the rate of TAN decrease) was positively correlated with the percentage of fast-twitch fibers (r = 0.92; P less than 0.001) and the ATP turnover rate (r = 0.75; P less than 0.001) but was not related to the muscle lactate content (r = 0.27; P greater than 0.05). Phosphocreatine decreased to 6.1 +/- 0.7 mmol/kg dm (range = 1-11) after contraction. It is concluded that during exercise activation of AMP deaminase in vivo occurs when a high ATP turnover rate is coupled with a low phosphocreatine level, muscle pH is of minor importance for direct activation of AMP deaminase in vivo, and increases in NH3 do not have an important influence on glycolysis.

AMP deaminase activity was inhibited in gill and muscle of Sarotherodon mossambicus, subjected to a week-long exposure to sublethal concentration of ambient ammonia. pH-dependent kinetic studies revealed decreased catalytic efficiency of the enzyme due to altered active site density and ionization pattern in the fish tissues at different phases of ammonia exposure.

Adenosine monophosphate (AMP) deaminase and 5'-nucleotidase, the two enzymes involved in the disposal of AMP, have been detected in different regions of normal rat brain and in animals subjected to heightened neuronal activity (leptazol-induced convulsions) and to depression of the central nervous system (CNS) by the administration of barbiturates. They have also been estimated in the CNS of animals subjected to anoxia or treated with lithium and ammonium salts. The AMP deaminase activity was found to be highest in cerebellum and lowest in cerebral cortex, while the 5'-nucleotidase activity was found to be highest in brain stem and lowest in cerebellum. The AMP deaminase activity was elevated in all the regions of brain during the preconvulsive and convulsive periods. The activity returned to normal during recovery. The activity of 5'-nucleotidase was found to be depressed in the preconvulsive and post-convulsive periods. The enzyme was also found to be depressed in all the three regions after the administration of barbiturates. Administration of lithium or ammonium salts of induction of anoxic states resulted in an increase in the activity of AMP deaminase in all the three regions of brain. These results are discussed in relation to the probable production of cyclic AMP and cyclic guanosine monophosphate (GMP) which may have depressive and excitatory roles, respectively, in brain. It appears that increased AMP deaminase activity is associated with increased neuronal activity while depression of 5'-nucleotidase activity is associated with conditions of decreased CNS excitability.

Publisher Summary This chapter describes the purification procedure of adenosine monophosphate (AMP) deaminase from human erythrocytes. AMP deaminase activity may also be measured by a spectrophotometric assay that records either the decrease in optical density at 265 nm as AMP is hydrolyzed or the increase in optical density at 240 nm. The enzyme migrates as an essentially single band in gel electrophoresis in sodium dodecyl sulfate (SDS) and has an estimated subunit molecule weight of 74,000. Partially purified AMP deaminase binds readily and reversibly to the inner (cytoplasmic) surface of isolated human erythrocyte membranes. Membrane-bound enzyme is less than 20% as active as enzyme fully dissociated from the membrane. These relationships have not been investigated with the fully purified enzyme preparations.

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.

Purified rat muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) binds tightly to rat myosin. The binding is abolished in the presence of low concentrations of various ligands. Pyrophosphate and GTP at concentrations as low as 0.1 micrometer were effective in abolishing the interaction between two proteins. Other nucleoside triphosphates were less effective than GTP and the concentrations required for 50% inhibition were approximately 0.3 to 0.7 micrometer. ADP and AMP are effective in inhibiting the interaction between two proteins, but they are less effective than the nucleoside triphosphates; 50% inhibition occurred at 34 micrometer with ADP and at 1 mM with AMP. Creatine phosphate and inorganic phosphate showed 50% inhibition at 5 to 6 mM. All of the compounds, which affected AMP deaminase activity, were effective in abolishing the interaction of the enzyme with myosin; however, the interaction-abolishing effects of the compounds are not parallel with their inhibitory effects on the deaminase activity. Although there exist three parental isozymes of AMP deaminase in the rat, all three enzymes interacted with myosin.

A method was developed to assay adenosine monophosphate (AMP) deaminase (AMP aminohydrolase, EC 3.5.4.6) in erythrocytes. Determination of AMP deaminase is complicated by the fact that, while AMP is disappearing under the influence of this enzyme, it is also being formed through the breakdown of adenosine triphosphate and adenosine diphosphate. We have found that hemolysis is followed by fairly rapid conversion of erythrocytic adenosine triphosphate and adenosine diphosphate to AMP. In this system, AMP deaminase activity was found to follow first order kinetics, the half-time being approximately 1 hr. at 37 C. The levels of the various intermediates were followed by ion-exchange chromatography to eliminate any complications or assumptions related to enzyme assays or substrate levels. Of the various additives used or under study in blood preservation, inorganic phosphate displayed a most remarkable inhibitory effect on AMP deaminase. This effect was related to the concentration of added phosphate. Adenine also inhibited the reaction. This inhibition was maximal at 1 mM and less at 0.5 or 2 mM. It appears that the ability of adenine to inhibit AMP deaminase plays an important role in its activity in preserving red blood cells. Inosine and dipyridamole (an adenosine deaminase inhibitor) appeared to have only slight effect on the activity of AMP deaminase.

A mutant clone of murine S49 cells, DTB6, was isolated in a single step from mutagenized wild type cells by virtue of its resistance to 1 mM thymidine and 1mM dibutyryl cyclic AMP. In comparative growth rate experiments, DTB6 cells were considerably less sensitive than parental cells to the growth inhibitory effects of thymidine and thymidine analogs, but surprisingly were equally sensitive to dibutyryl cyclic AMP. Conversely, DTB6 cells were much more sensitive to the cytotoxic effects of either adenine or adenosine-EHNA. This supersensitivity of DTB6 cells to growth inhibition by adenine could be ameliorated by the addition of hypoxanthine to the culture medium. The complex growth phenotype of the mutant cells could be attributed to a 60% deficiency in AMP deaminase activity and a complete absence of thymidine kinase activity in the mutant cells. Revertants of DTB6 cells possessed wild type levels of AMP deaminase activity but remained deficient in thymidine kinase activity, while another revertant of DTB6 cells expressed 11% of the wild type thymidine kinase level but did not perceptibly change its AMP deaminase activity. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism.