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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.

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 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.

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.

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.

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.

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.

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.

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.

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.

The mechanism of activation of glutamine production by the hindlimb during diabetic ketoacidosis (DKA) was investigated in rats. Muscle glutamine production was estimated to account for over 90% of the total glutamine produced by the hindlimb. DKA produced significant increases in the concentrations of NH4+ and IMP in hindlimb muscles, suggesting that AMP deaminase is activated by DKA. NH4Cl- and HCl-induced acidosis did not produce these changes, indicating either that acidosis itself is not the stimulus for increased AMP deaminase activity or that the more severe degree of acidosis accompanying DKA is necessary for activation. Muscle glutamine concentrations were depressed in DKA. Experiments with isolated epitrochlearis muscle showed that the transport and permeability properties of the muscle cells (as judged by uptake and release of alpha-aminoisobutyrate and glutamine) were not altered by DKA. However, glutamine uptake by muscle cells was significantly inhibited by L-leucine, the concentration of which, along with other branched-chain amino acids, is markedly elevated in DKA.

The role of AMP deaminase (EC 3.5.4.6) reaction in the stimulation of the regulatory enzymes of glycolysis was investigated using permeabilized yeast cells. 1) The addition of polyamine activated AMP deaminase in situ, resulting in the subsequent increase in ammonium production, which can stimulate the activity of 6-phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40). 2) Zn2+ inhibited AMP deaminase activity, followed by a decrease in ammonium ion concentration which reduced the activity of phosphofructokinase. 3) Polyamine and Zn2+ did not activate or inhibit directly the activity of phosphofructokinase and pyruvate kinase. 4) A simple Michaelis-Menten relationship was observed between the various levels of ammonium ion and of fructose 1,6-biphosphate formed in situ, indicating that phosphofructokinase activity or glycolytic flux was dependent upon the level of ammonium produced through the action of AMP deaminase. 5) The increase in Pi concentration resulted in the decreased magnitude of activation by NH4+ and marked stimulation by Pi itself of phosphofructokinase, and further reduced the production of NH4+ through the inhibition of AMP deaminase, suggesting that phosphofructokinase activity may not be regulated by the level of NH4+ but by Pi concentration under conditions of increased Pi levels. The AMP deaminase-ammonium system shows a regulatory function in glycolysis of yeast cells in the presence of physiological Pi levels, whereas glycolysis may be principally controlled by Pi level under the conditions of elevated Pi concentration. Polyamines may play a part in the stimulation of glycolysis through the elevated level of ammonium ion under the conditions of increased ATP utilization during cell proliferation, and can participate in the catabolic processes as well as anabolic processes through the stimulation of the AMP deaminase-ammonium system.

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

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.

Erythrocytes from 11 patients with presumptive diagnoses of transient erythroblastopenia of childhood were evaluated retrospectively (six) or prospectively (five) for a possible relationship between erythrocyte adenosine 5′-monophosphate aminohydrolase, adenylic acid deaminase (AMP deaminase) activity and intracellular concentrations of adenine nucleotides. Older red blood cell (RBC) cohorts in these patients consistently exhibited significantly decreased activities of AMP deaminase (approximately 5% to 70% of normal control mean) in association with increased concentrations (up to threefold) of adenosine triphosphate (ATP) and total adenine nucleotides. We postulate that the latter is a direct consequence of the former, since diminishing AMP deaminase activity in aging cells should reduce the drain on the adenine nucleotide pool imposed by irreversible deamination of AMP to inosine 5′-monophosphate. Consistent reductions in AMP deaminase activity indicate that this enzyme should also serve as a reliable marker of mean RBC age useful in diagnostic confirmation of transient erythroblastopenia. The observed increases in ATP and total adenine nucleotides in older RBCs require a reevaluation of the traditional view that age-related losses of these compounds mediate the ultimate demise of senescent erythrocytes. Similar alterations in the balance of degradative and salvage pathways in RBC nucleotide metabolism may also underlie certain cases of so-called “high ATP syndrome.”

AMP deaminase (EC 3. 5. 4. 6) is a key enzyme in the adenine nucleotide metabolism of human erythrocytes. ATP activates the enzyme, while 2, 3-bisphosphoglycerate inhibits it. Complexes of magnesium and these organic phosphate compounds are not effectors of AMP deaminase. The AMP deaminase activity in the physiological range of AMP was calculated from AMP-saturation curves which were drawn by measuring the activity under simulated intracellular conditions. Since human erythrocytes have a high level of AMP deaminase, the enzyme activity must be repressed to a low level, which accounts for the half-life of adenine nucleotides in the erythrocyte. Although it is evident from the experimental results that 2, 3-bisphosphoglycerate plays an important role in the repression of the activity of this enzyme, the in vivo rate of turnover of adenine nucleotides can not be explained by the inhibitory effect of 2, 3-bisphosphoglycerate alone.

Myocardial infarctions were induced in rats by injecting isoproterenol at 800 mg/kg body weight in divided doses 24 h apart. Both DNA synthesis and AMP deaminase activity were stimulated by this regimen, with peak stimulation of both occurring about 48 h after the first injection of isoproterenol. Greater increases in DNA synthesis and enzyme activity were observed in ventricular areas than in auricular areas in correlation with the extent of damage during infarction. AMP deaminase activity also increased as the dose of isoproterenol increased up to a concentration of 800 mg/kg body weight in divided doses. Kinetic studies on AMP deaminase in heart extracts from control or isoproterenol-injected rats showed the enzyme to be allosteric and its activity was modulated by alkali metal ions, ATP and GTP. In the presence of alkali metal ions or ATP, velocity vs. substrate plots adhered to simple first-order Michaelis-Menten formulation; and activity in heart extracts from isoproterenol-injected rats exceeded activity in controls by 2 to 3-fold. Therefore, it appeared that increased enzyme activity was associated with increases in enzyme amount.

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.

When ATP was maintained at a high concentration by respiration or glycolysis (or both) in ascites tumor cells incubated in vitro, the adenine nucleotide quotient, (ATP) (AMP)/(ADP)2, was several-fold higher than the adenylate kinase equilibrium value. In such cells with high ATP:ADP ratios, incorporation of 32Pi into the γ-phosphoryl position of ATP was higher than that into the β position. Subsequent addition of excess inorganic phosphate to the medium of these cells effected more rapid loss of radioactivity from the γ-phosphoryl position. These results indicate that under conditions of rapid ATP generation the adenylate kinase fails to phosphorylate AMP as rapidly as necessary to maintain equilibrium. A number of conditions that retarded ATP generation, as shown by the lowering of the ATP:ADP ratio, also decreased the adenine nucleotide quotient to values in the range of the adenylate kinase equilibrium. These are the addition of 2-deoxyglucose, which depleted the cellular inorganic phosphate content and hence inhibited oxidative phosphorylation, and the addition of glyoxylate, which blocked the tricarboxylic acid cycle. Factors that reversed these effects, glucose and l-glutamate, respectively, also restored the high ATP:ADP ratio and the nonequilibrium state of the adenylate kinase reaction. Incubation of the cells with adenosine, inosine, or l-glutamine increased the total adenine nucleotide content. 2-Deoxyglucose induced rapid depletion of the adenine nucleotide pool, probably as a result of enhanced deamination of AMP in the inorganic phosphate-deficient cells. Although glyoxylate increased the AMP level, it also increased the cellular inorganic phosphate and thus had little effect on the total adenine nucleotide content. These results advance the conclusion that inorganic phosphate levels are important in the control of AMP deaminase activity in the intact cell.

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.

Five cases of a new disease presented with muscular weakness or cramping after exercise; three of the cases also had an elevated serum creatine phosphokinase. Muscle biopsies were histologically normal but lacked adenylate deaminase by stain and solution assay, while the erythrocyte isozyme was normal. A clinical diagnostic test has been developed, and the human enzyme was separated by acrylamide-gel electrophoresis.

AMP deaminase isoforms from human skeletal muscle can be separated chromatographically [Kaletha, Spychała & Nowak (1987) Experientia 43, 440-443]. In adult tissue nearly all the AMP deaminase activity was eluted from phosphocellulose with 0.75 M-KCl (‘adult’ isoform), and the remaining activity could be eluted with 2.0 M-KCl. Conversely, most of the AMP deaminase activity from 11-week-old fetal tissue was eluted from phosphocellulose with 2.0 M-KCl (‘fetal’ isoform). In the present paper the kinetic and regulatory properties of AMP deaminase extracted from 11- and 16-week-old fetal skeletal muscle are reported. The two isoforms from 11-week-old human fetus differed distinctly in these properties. The ‘fetal’ isoform had about 5-fold higher half-saturation constant (S0.5) value than the ‘adult’ form. It was also more sensitive to the influence of some important regulatory ligands (ADP, ATP and Pi), and exhibited a different pH/activity profile. The ‘adult’ isoform of AMP deaminase from fetal muscle and the enzyme from mature muscle possessed similar kinetic and regulatory properties. This isoform seems not to be subject to any major modifications during further ontogenesis. This is not true, however, for the ‘fetal’ isoform. In the muscle of 16-week-old human fetus, the ‘fetal’ isoform showed a peculiar, biphasic, type of substrate-saturation kinetics. This phenomenon may reflect appearance of the next, developmentally programmed, isoform of human skeletal-muscle AMP deaminase.

Activity levels of AMP deaminase and adenosine deaminase have been studied in hepatopancreas, foot and mantle tissues ofPila globosa with reference to starvation and aestivation. The activity levels of both enzymes were decreased in all the tissues of aestivating snails while in starved animals AMP deaminase activity was increased, in contrast to the decreased adenosine deaminase activity.

The histochemical method for AMP deaminase confirms that rat slow-twitch fibres contain less AMP deaminase activity than fast-twitch fibres. However, the enzyme appears similarly localized in all fibre types.

The deficiency was first found by Ogasawara et al /BBRC 1984, 122, 1344 - 1349, Hum.Genet. 1987, 75, 15 - 18/ to occur in Japan, Seoul and Taipei as one heterozygote in about 30 of the population. In order to find the frequency of the defect in the european area, 410 blood samples have been collected from the blood donors in the county blood center in Gdansk /Poland/. The mean AMP deaminase activity in the erythrocytes was found to be 8.3 μmole ammonia/min per g hemoglobin, which is about 30% less than found in Japan but exactly the same which was found in U.S.A. by Campbell et al. /Clin.Chim Acta 1977, 79, 379 - 385/. Among the 410 individuals investigated 26 displayed half or less of the mean erythrocyte AMP deaminase activity. This could mean that the frequency of the mutant gene is one heterozygote in about 16 of the population in Poland. One case of a complete deficiency of the erythrocyte AMP deaminase activity was found in the family member of one of the heterozygotes. All the deficient subjects appeared to be perfectly healthy, there were no evidence of either anaemia or hemolysis.

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.