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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.

Adenyl nucleotide levels and activity of AMP catabolism enzymes in the cytosolic liver fraction of rats with acetaminophen-induced hepatitis have been studied under different dietary protein regimens. It was found that in animals with toxic hepatitis maintained on a diet rich in protein the ATP and ADP levels decreased, while AMP levels were similar to those in control animals. At the same time, in the cytosolic liver fraction of rats with acetaminophen-induced hepatitis kept under conditions of protein deficiency, ATP and AMP pools were depleted. Changes in the adenyl nucleotides content were accompanied by altered activity of AMP catabolism enzymes, such as 5′-nucleotidase and AMP deaminase. It was found that in rats with toxic hepatitis that were fed a complete diet, AMP deaminase activity increased in comparison to the control level along with 5′-nucleotidase stimulation. At the same time, in protein-restricted rats with toxic liver damage, AMP deaminase activity decreased, while 5′-nucleotidase activity was elevated in comparison to control values. These results indicate depletion of energy sources in the liver cells of rats with acetaminophen-induced hepatitis that were fed a low-protein diet. The observed changes in the activity of AMP catabolism enzymes may be considered as one of the mechanisms that regulate the cellular energy function.

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.

SummaryInhibition of AMP deaminase (AMPD) holds the potential to elevate intracellular adenosine and AMP levels and, therefore, to augment adenosine signaling and activation of AMP-activated protein kinase (AMPK). To test the latter hypothesis, novel AMPD pan inhibitors were synthesized and explored using a panel of in vitro, ex vivo, and in vivo models focusing on confirming AMPD inhibitory potency and the potential of AMPD inhibition to improve glucose control in vivo. Repeated dosing of selected inhibitors did not improve glucose control in insulin-resistant or diabetic rodent disease models. Mice with genetic deletion of the muscle-specific isoform Ampd1 did not show any favorable metabolic phenotype despite being challenged with high-fat diet feeding. Therefore, these results do not support the development of AMPD inhibitors for the treatment of type 2 diabetes.

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.

Vertebrates have acidic and basic isozymes of adenylosuccinate synthetase, which participate in the first committed step of de novo AMP biosynthesis and/or the purine nucleotide cycle. These isozymes differ in their kinetic properties and N-leader sequences, and their regulation may vary with tissue type. Recombinant acidic and basic synthetases from mouse, in the presence of active site ligands, behave in analytical ultracentrifugation as dimers. Active site ligands enhance thermal stability of both isozymes. Truncated forms of both isozymes retain the kinetic parameters and the oligomerization status of the full-length proteins. AMP potently inhibits the acidic isozyme competitively with respect to IMP. In contrast, AMP weakly inhibits the basic isozyme noncompetitively with respect to all substrates. IMP inhibition of the acidic isozyme is competitive, and that of the basic isozyme noncompetitive, with respect to GTP. Fructose 1,6-bisphosphate potently inhibits both isozymes competitively with respect to IMP but becomes noncompetitive at saturating substrate concentrations. The above, coupled with structural information, suggests antagonistic interactions between the active sites of the basic isozyme, whereas active sites of the acidic isozyme seem functionally independent. Fructose 1,6-bisphosphate and IMP together may be dynamic regulators of the basic isozyme in muscle, causing potent inhibition of the synthetase under conditions of high AMP deaminase activity.

Postmortem energy metabolism drives hydrogen accumulation in muscle and results in a fairly constant ultimate pH. Extended glycolysis results in adverse pork quality and may be possible with greater adenonucleotide availability postmortem. We hypothesized that slowing adenonucleotide removal by reducing AMP deaminase activity would extend glycolysis and lower the ultimate pH of muscle. Longissimus muscle samples were incorporated into an in vitro system that mimics postmortem glycolysis with or without pentostatin, an AMP deaminase inhibitor. Pentostatin lowered ultimate pH and increased lactate and glucose 6-phosphate with time. Based on these results and that AMPK γ3 R200Q mutated pigs (RN − ) produce low ultimate pH pork, we hypothesized AMP deaminase abundance and activity would be lower in RN − muscle than wild-type. RN − muscle contained lower AMP deaminase abundance and activity. These data show that altering adenonucleotide availability postmortem can extend postmortem pH decline and suggest that AMP deaminase activity may, in part, contribute to the low ultimate pH observed in RN − pork.

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a Km of 2 mM for 5′-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the Km of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD+, whereas, the Km was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in Vmax with no change in Km. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.

AMPD1 genotype, relative fiber type composition, training status, and gender were evaluated as contributing factors to the reported variation in AMP deaminase enzyme activity in healthy skeletal muscle. Multifactorial correlative analyses demonstrate that AMPD1 genotype has the greatest effect on enzyme activity. An AMPD1 mutant allele frequency of 13.7 and a 1.7% incidence of enzyme deficiency was found across 175 healthy subjects. Homozygotes for the AMPD1 normal allele have high enzyme activities, and heterozygotes display intermediate activities. When examined according to genotype, other factors were found to affect variability as follows: AMP deaminase activity in homozygotes for the normal allele exhibits a negative correlation with the relative percentage of type I fibers and training status. Conversely, residual AMP deaminase activity in homozygotes for the mutant allele displays a positive correlation with the relative percentage of type I fibers. Opposing correlations in different homozygous AMPD1 genotypes are likely due to relative fiber-type differences in the expression of AMPD1 and AMPD3 isoforms. Gender also contributes to variation in total skeletal muscle AMP deaminase activity, with normal homozygous and heterozygous women showing only 85–88% of the levels observed in genotype-matched men.

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.

Using AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) purified from rabbit left-ventricular heart tissue, we report direct investigation of the potential for cardiac AMP deaminase activity to be regulated by kinase-mediated phosphorylation. Rabbit heart AMP deaminase served as a substrate for Ca2+/phospholipid-dependent protein kinase (protein kinase C; PKC) exclusively; no other mammalian protein kinase phosphorylated the enzyme. PKC-dependent AMP deaminase phosphorylation was rapid, linear with respect to time and the concentrations of PKC and AMP deaminase in the reaction, and inhibitable by staurosporine. Upon phosphorylation, the apparent Km of cardiac AMP deaminase decreased from 5.6 mM to 1.2 mM, without effect on the Vmax. Whether phosphorylated or not, rabbit heart AMP deaminase was inhibited by 1.0 mM GTP, which decreased the Vmax. by approximately 50% in each case. PKC-dependent phosphorylation of cardiac AMP deaminase did not alter the enzyme's allosterism toward millimolar ATP or ADP: both nucleotides at 1.0 mM concentration decreased the apparent Km to approximately 0.5 mM. Treatment of cardiac phospho-AMP deaminase with either the protein phosphatase calcineurin or alkaline phosphatase generated a dephosphorylated form which displayed molecular and kinetic properties identical with those of the originally isolated enzyme. These data raise the possibility that a phosphorylation-dephosphorylation mechanism may regulate flux through AMP deaminase in the heart under pathological conditions, such as myocardial ischaemia, characterized by PKC activation and adenylate depletion.

The mechanism for how metformin activates AMPK (AMP-activated kinase) was investigated in isolated skeletal muscle L6 cells. A widely held notion is that inhibition of the mitochondrial respiratory chain is central to the mechanism. We also considered other proposals for metformin action. As metabolic pathway markers, we focused on glucose transport and fatty acid oxidation. We also confirmed metformin actions on other metabolic processes in L6 cells. Metformin stimulated both glucose transport and fatty acid oxidation. The mitochondrial Complex I inhibitor rotenone also stimulated glucose transport but it inhibited fatty acid oxidation, independently of metformin. The peroxynitrite generator 3-morpholinosydnonimine stimulated glucose transport, but inhibited fatty acid oxidation. Addition of the nitric oxide precursor arginine to cells did not affect glucose transport. These studies differentiate metformin from inhibition of mitochondrial respiration and from active nitrogen species. Knockdown of adenylate kinase also failed to affect metformin stimulation of glucose transport. Hence, any means of increase in ADP appears not to be involved in the metformin mechanism. Knockdown of LKB1, an upstream kinase and AMPK activator, did not affect metformin action. Having ruled out existing proposals, we suggest a new one: metformin might increase AMP through inhibition of AMP deaminase (AMPD). We found that metformin inhibited purified AMP deaminase activity. Furthermore, a known inhibitor of AMPD stimulated glucose uptake and fatty acid oxidation. Both metformin and the AMPD inhibitor suppressed ammonia accumulation by the cells. Knockdown of AMPD obviated metformin stimulation of glucose transport. We conclude that AMPD inhibition is the mechanism of metformin action.

1. 1. Replacement of fetal calf serum and chicken embryo extract by Ultroser G and rat brain extract during the proliferation phase resulted in a higher maturation grade of cultured rat muscle cells after 7 days of differentiation, on base of the percentage of the muscle specific isoenzyme of creatine kinase (CK-MM). 2. 2. Furthermore, the activities of creatine kinase, citrate synthase, cytochrome c oxidase and hexokinase were significantly higher. 3. 3. Compared to the enzyme activities in m. quadriceps of 10 day-old rat and m. quadriceps m. soleus and m. extensor digitorum longus of young adult rats, the metabolic capacity of cultured myotubes most closely resembles that of the first muscle. 4. 4. Paralysis with tetrodotoxin caused a slight decrease of the creatine kinase activity and the percentage of CK-MM of cultured myotubes and an increase of the activities of hexokinase, phosphorylase and AMP deaminase. 5. 5. Electrical stimulation performed at different frequencies and time periods had no effect on the enzyme activities of cultured rat muscle cells. 6. 6. Only the AMP deaminase activity was decreased after intense electrical stimulation.

Cytosolic enzymes AMP deaminase and adenosine deaminase (ADA) catalyze AMP and adenosine deamination, constitute rate-limiting steps of adenine nucleotide catabolism and play important roles in cellular energy metabolism. In this study, AMP deaminase and ADA activities of rat liver, neocortex, cerebellum, striatum and hippocampus were investigated in acute ammonia intoxication and subacute CCl(4)-induced hepatitis. Activities of both AMP deaminase and ADA in the liver were elevated by 2.4-4.2-fold (p

AMP diaminase from normal and diabetic rat hearts was separated on cellulose phosphate and quantified by HPLC. From soluble fractions three different AMP deaminase activities, according to KCl elution from cellulose phosphate and percent of total activity were: 170 mM (85%), 250 mM (8%) and 330 mM (7%) KCl. The AMP deaminase activity which eluted with 170 mM KCl was resolved to two distinct peaks by HPLC anionic exchange. After 4 weeks of diabetes the heart enzyme profile changed to: 170 mM (10%), 250 mM (75%) and 330 mM (15%). Once purified the four activities were kinetically distinct: 170 mM KCl cytosolic, AMP K m = 1.78, simulated by ATP, GTP, NADP and strongly inhibited by NAD; 170 mM KCl mitochondria, AMP K m = 17.9, stimulated by ATP, ADP; 250 mM KCl isozyme, AMP K m = 0.66, stimulated by ADP; and 330 mM KCl isozyme, AMP K m = 0.97, inhibited by ATP, NAD(P).

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC(50) = 0.5 micro M. AMPDI was much less effective with isolated cardiomyocytes (IC(50) = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.

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

We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability.

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.

SUMMARYA cDNA coding for a protein with significant similarity to adenosine deaminase (ADA) was found while randomly sequencing a cDNA library constructed from salivary gland extracts of adult female Culex quinquefasciatus. Prompted by this result, we found high ADA activities in two culicine mosquitoes, Culex quinquefasciatus and Aedes aegypti, but not in the anopheline Anopheles gambiae. Homogenates from Culex quinquefasciatus also have an AMP deaminase activity that is three times greater than the ADA activity, whereas in Aedes aegypti the AMP deaminase activity is less than 10% of the ADA activity. Evidence for secretion of ADA during blood feeding by Aedes aegypti includes the presence of ADA activity in warm solutions probed through a membrane by mosquitoes and in serotonin-induced saliva and a statistically significant reduction in the levels of the enzyme in Aedes aegypti following a blood meal. We could not demonstrate, however, that C. quinquefasciatus secrete ADA in their saliva. Male Aedes aegypti and C. quinquefasciatus, which do not feed on blood, have less than 3% of the levels of ADA found in females. We propose that ADA activity in A. aegypti may help blood feeding by removing adenosine, a molecule associated with both the initiation of pain perception and the induction of mast cell degranulation in vertebrates, and by producing inosine, a molecule that potently inhibits the production of inflammatory cytokines. The role of salivary ADA in Culex quinquefasciatus remains unclear.

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.

Alterations in the competency of the creatine kinase system elicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular and kinetic changes in AMP deaminase from skeletal muscles of mice deficient in either cytosolic creatine kinase alone (M-CK−/−) or also deficient in mitochondrial creatine kinase (CK−/−) compared with wild type. We found that predominantly fast-twitch muscle, but not slow-twitch muscle, from both M-CK−/−and CK−/−mice had much lower AMP deaminase; the quantity of AMP deaminase detected by Western blot was correspondingly lower, whereas AMP deaminase-1 ( AMPD1) gene expression was unchanged. Kinetic analysis of AMP deaminase from mixed muscle revealed negative cooperativity that was significantly greater in creatine kinase deficiencies. Treatment of AMP deaminase with acid phosphatase abolished negative cooperative behavior, indicating that a phosphorylation-dephosphorylation cycle may be important in the regulation of AMP deaminase.

Few studies examine ammonia and amino acid metabolism in response to endurance training. Trained humans generally experience less increase in plasma ammonia during either prolonged or intense exercise. This is probably a reflection of reduced ammonia production and release from the active muscle; it could be a reflection of decreased AMP deaminase activity, decreased glutamate dehydrogenase activity, and/or increased alanine and glutamine formation. Little is known regarding the associated enzyme systems in humans, but in experiments with animal models, aerobic training decreases AMP deaminase and increases the enzymes of amino acid transamination and oxidation.

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.

Using an in situ assay for analyzing AMP deaminase activity in isolated adult rat ventricular myocytes, we have shown that IMP production is stimulated approximately twofold in cardiac cells incubated with 10 microM adenosine. This effect of adenosine was not blocked by the adenosine A1-receptor antagonist 8-cyclophenyl-1,3-dipropylaxanthine (0.01-1 microM) except at a concentration (100 microM) that may inhibit adenosine transport. Similarly, in situ AMP deaminase activity was not enhanced by treatment with the specific adenosine A1-receptor agonists N6-phenylisopropyl adenosine or cyclopentyladenosine, nor was it sensitive to prior treatment of cells with pertussis toxin. The nucleoside transport blockers S-4-nitrobenzyl-6-thioinosine, dipyridamole, and papaverine inhibited adenosine-induced increases in IMP production by 75-85%, suggesting an intracellular site of action. Modulation of enzyme activity via the transmethylation pathway could not be implicated since incubation of cardiac cells under conditions known to elevate intracellular S-adenosyl-L-homocysteine had no demonstrable effect on AMP deaminase. Furthermore, a direct allosteric effect of adenosine on the partially purified rat cardiac enzyme was not observed. The results indicate that intracellular adenosine modulates rat cardiac AMP deaminase by an unknown mechanism.

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.

In Chinese hamster fibroblasts, we previously detected an expressed gene located near the AMP deaminase gene. This gene was named Y1. Upon selection for resistance to coformycin, an inhibitor of AMP deaminase activity, both genes were amplified in several mutants. We have determined the complete nucleotide sequence of Y1 cDNA and identified the Y1 gene as a mu class glutathione S-transferase gene by comparison with sequences present in a data bank. Accordingly, Y1-amplified mutants express an increased glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene; this activity, as well as the abundance of the corresponding RNA, appears, however, to reach a limit despite further increase in the Y1 gene copy number during successive amplification steps. Southern blot experiments showed that Y1 belongs to a multigene family, all or part of which has been amplified in mutant lines. These data provide a method to amplify and to overexpress the mu class of the glutathione S-transferase gene family on the basis of its linkage with the AMP deaminase gene.

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 kinetic and regulatory properties of homogeneous AMP deaminase from rat skeletal muscle have ben examined with particular emphasis on (a) the role of potassium ions in the expression of these properties and (b) the role of the adenylate energy charge in the regulation of AMP deaminase activity. Although the enzyme has an absolute requirement for K+, the concentration required for maximum activation is dependent on the concentration of substrate. At saturating levels of AMP (greater than or equal 2.0 mM) maximum activation is observed with 25 mM KCl, whereas at lower substrate concentrations (0.2 mM) approximately 50 mM KCl is needed for maximum activation. Conversely, the response of enzyme activity ot increasing levels of substrate is dependent on the level of potassium ions. At substrating concentrations of K+, the saturation curve for AMP is highly sigmoidal (nh=3.2) whereas at higher KCl concentrations, the apparent cooperativity between AMP sites is almost completely abolished (nh=1.5). The inhibition by a number of phosphorylated metabolites, including ATP, GTP, creatine-P, and P1, is likewise sensitive to the concentration of K+. These results suggest that a significant amount of interaction between K+ sites and both substrate and effector sites is required for the expression of the catalytic and regulatory properties of the enzyme. The specific effects of ATP, creatine-P, and P1 on the parameters of Km and Vmax indicate that each of these profile of AMP deaminase activity generated in response to variations in the adenylate energy charge shows that within the physiological range of energy charge (0.75 to 0.95), the activity increases linearly with decreasesing energy charge and is insensitive to both the total adenylate pool size and the presence of P1 and creatine-P. These data suggest that the most important factor in the regulation of AMP deaminase activity is the state of the energy charge rather than the absolute concentrations of the individual effectors.

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.

Fluorescent antibody staining experiments with both isolated myofibrils and muscle fibers grown in culture show that AMP deaminase is bound to the myofibril in the A band. The strongest staining occurs at each end of the A band. The approximate width of the fluorescent stripes and their relation to the A band remains constant as a function of sarcomere length. Removal of enzyme from the myofibrils leads to loss of staining, and readdition of purified enzyme restores the original staining pattern. A histoenzymatic method for the detection of AMP deaminase activity in cultured fibers gives comparable localization. The results are consistent with the previous observation (Ashby, B. and C. Frieden. 1977.J. Biol. Chem. 252:1869--1872) that AMP deaminase forms a tight complex in solution with subfragment-2 (S-2) of myosin or with heavy meromyosin (HMM).

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.

Myoadenylate deaminase is the muscle-specific isoform of AMP deaminase (EC 3.5.4.6), an enzyme which plays a special role in energy metabolism in skeletal muscle. A 2.3-kilobase cDNA encoding this enzyme has been cloned from a lambda gt10 library prepared from rat skeletal muscle using oligonucleotide probes designed from AMP deaminase peptide sequences. This cDNA was sequenced, and the amino acid sequence of this isoform of AMP deaminase was deduced. Sequences homologous to this cDNA are identified in the genome of eukaryotes as diverse as yeast and man. Tissue-specific expression of a 2.5-kilobase AMP deaminase transcript is demonstrated, and the abundance of this transcript as well as the 80-kDa adult, muscle-specific peptide of AMP deaminase increase in parallel during postnatal skeletal muscle development. In the adult animal, the abundance of this transcript and AMP deaminase activity are differentially expressed in various skeletal muscle fiber types. We conclude that AMP deaminase sequences have been highly conserved during evolution, and in mammals there is developmental and tissue-specific control of expression of this gene.

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.