AMP Deaminase Activity Research Articles

Enzymes involved in purine metabolism--a review of histochemical localization and functional implications.

Many enzymes are involved in the biosynthesis, interconversion, and degradation of purine compounds. The exact function of these enzymes is still unknown, but they seem to play important roles other than in purine metabolism. To elucidate their functional roles, it is imperative to clarify their tissue distribution at the cellular or subcellular level. The present review summarizes the currently available information about their histochemical localization and proposed functions. In general, 5'-nucleotidase has been considered as a marker enzyme for the plasma membrane, and is considered to be a key enzyme in the generation of adenosine, a potential vasodilator. However, from its wide range of localization in tissues it is also considered to be related to the membrane movement of cells in the transitional epithelium, cellular motile response, transport process, cellular growth, synthesis of fibrous protein and calcification, lymphocyte activation, neurotransmission, and oxygen sensing mechanism. Adenosine deaminase (ADA) is present in all tissues in mammals. Although the main function of ADA is the development of the immune system in humans, it seems to be associated with the differentiation of epithelial cells and monocytes, neurotransmission, and maintenance of gestation. Purine nucleoside phosphorylase (PNP) is generally considered as a cytosolic enzyme, but recently, mitochondrial PNP, a different protein from cytosolic PNP, was reported. PNP is also widely expressed in human tissues. It is found in most tissues of the body, but the highest activity is in peripheral blood granulocyte and lymphoid tissues. It is also related to the development of T-cell immunity in humans as is ADA. Moreover, its contribution to centriole replication and/or regulation of microtubule assembly has been suggested. Immunohistochemical localization of xanthine oxidase has been reported in various tissues from various animal species. Xanthine oxidase has been suggested to be involved in the pathogenesis of post-ischemic reperfusion tissue injury through the generation of reactive oxygen species, while the extensive tissue localization of xanthine dehydrogenase/oxidase suggests several other roles for this enzyme, including a protective barrier against bacterial infection by producing either superoxide radicals or uric acid. Furthermore, an involvement in cellular proliferation and differentiation has been suggested. Urate oxidase is generally considered a liver-specific enzyme, except for bovines which possess this enzyme in the kidney. Urate oxidase is exclusively located in the peroxisomes of fish, frogs, and rats, but was lost in birds, some reptiles, and primates during evolution. A histochemical demonstration of allantoin-degrading enzymes has not been performed, but these enzymes have been located in peroxisomes by sucrose density gradient centrifugation. AMP deaminase activity is higher in skeletal muscle than in any other tissues. AMP deaminase may be involved in a number of physiological processes, such as the conversion of adenine nucleotide to inosine or guanine nucleotide, stabilizing the adenylate energy charge, and the reaction of the purine nucleotide cycle. There are three distinct isozymes (A, B, C) with different kinetic, physical, and immunological properties. Isozymes A, B, C have been isolated from muscle, liver (kidney), and heart tissue, respectively. In the muscle, AMP deaminase isozymes exist in a different part, suggesting a multiple functional role of this enzyme. High hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity is found in some regions of a normal adult human brain. However, very little is known regarding the histochemical tissue localization of HGPRT. Immunohistochemical localization of its developmental expression suggests that HGPRT may not be essential for purine nucleotide supplement in the segmentation of brain cells, but may play a significant role in the developing hippocampus.

Regulation of AMP Deaminase by Phosphoinositides

AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K(i) of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/K)X(n)(R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo.

AMP deaminase in piglet cardiac myocytes: effect on nucleotide metabolism during ischemia.

The purpose of this study was to examine in situ regulation of AMP deaminase in newborn piglet cardiac myocytes and to determine its role in nucleotide metabolism during ischemia. When a rapid deenergization paradigm was used to assay AMP deaminase, enzyme activity depended on the hormonal and metabolic status of cells just before deenergization. Inosine 5'-monophosphate (IMP) formation was increased 150% in deenergized myocytes pretreated with phorbol 12-myristate 13-acetate (PMA; EC50 = 4.7 x 10(-8) M). This effect was 90% blocked with the protein kinase C (PKC) inhibitor staurosporine. In addition, the beta-adrenergic agonist isoproterenol stimulated AMP deaminase activity (EC50 = 1.5 x 10(-8) M), and IMP formation was directly correlated to intracellular cAMP levels (r2 = 0.9). Furthermore, adenosine increased IMP formation, whereas nonrespiring, glycolyzing piglet myocytes had reduced AMP deaminase activity. Pretreatment of perfused piglet hearts with adenosine, but not PMA, before exposure to global ischemia resulted in enhanced conversion of AMP to IMP during the ischemic period. Similar results were obtained in piglet myocytes preincubated with adenosine or PMA before exposure to simulated ischemia. These results may be relevant to the preconditioning phenomenon.

Isolation and regulation of piglet cardiac AMP deaminase.

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.

Product activation of human erythrocyte AMP deaminase

IMP was found to activate AMP deaminase in crude glucose-depleted human erythrocyte lysates. Activation of the enzyme by IMP is due to prevention of the inhibitory effect of inorganic phosphate. At 1 mM AMP and 2–3 mM phosphate the addition of 2–5 mM IMP accelerates the AMP deamination two to three times.

Genetic and other determinants of AMP deaminase activity in healthy adult skeletal muscle.

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.

Cyanide exposure affects the production and excretion of ammonia by the mudskipper Boleophthalmus boddaerti

The concentrations of ammonia in the plasma of the mudskipper Boleophthalmus boddaerti exposed to cyanide for 1–6 days were significantly greater than the respective values of the controls. This was due to an increase in the production of NH 3 in the muscle and an increase in the retention of NH 3 and/or NH 4 + in the blood of the cyanide-exposed fish when compared to controls. Cyanide exposure significantly increased the specific activity of muscle AMP deaminase. Since adenylosuccinate synthetase and lyase were also present in the muscle, exposure to cyanide might increase the production of NH 3 from the catabolism of purine nucleotides. B. boddaerti exposed to cyanide excreted significantly less ammonia than the control fish. Results indicate changes in the permeability of the epithelial surfaces of the cyanide-exposed fish to NH 3 and/or NH 4 +. Since the tissues and organs needed time to activate the inducible cyanide detoxification mechanisms, the increase in the production of NH 3 might be an important defensive mechanism for B. boddaerti during the early phase of cyanide exposure.

Structural–Functional Relationships in Pig Heart AMP-Deaminase in the Presence of ATP, Orthophosphate, and Phosphatidate Bilayers

The secondary structure of pig heart AMP-deaminase (AMP-d) in the absence and in the presence of orthophosphate or dioleoyl phosphatidic acid (DOPA) or ATP was investigated by FT-IR spectroscopy. While the latter substance activates the enzyme, orthophosphate is a well-known negative allosteric effector and DOPA exerts a noncompetitive inhibition on AMP-deaminase. Small changes in the secondary structure of AMP-d were induced by the above mentioned substances. Only DOPA reduced the thermal stability of AMP-d and avoided protein intermolecular interactions suggesting structural–functional relationships in AMP-d in the presence of the above substances and a possible role of phosphatidic acid in the subtle regulation of AMP-d activity by temporary binding of the enzyme to cellular membranes.

Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice.

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.

Metabolite accumulation increases adenine nucleotide degradation and decreases glycogenolysis in ischaemic rat skeletal muscle.

Adenine nucleotides and glycogen are degraded in skeletal muscle during no-flow ischaemia. Past investigations have ascribed these metabolic changes to the severe energetic stress which arises with the removal of exogenous substrates (principally oxygen). We tested this hypothesis by measuring the high-energy phosphagen and glycogen contents of stimulated rat hindlimb muscles (1 twitch s-1) prior to and following 40 min of no-flow ischaemia or hypoxic perfusion without glucose (PaO2 = 4.6 +/- 0.1 torr, plasma glucose = 0.3 +/- 0.1 mmol L-1). Both experimental protocols eliminated exogenous substrate supply; however, the maintenance of flow during hypoxic perfusion ensured the removal of metabolic by-products. A period of forty minutes of skeletal muscle ischaemia was characterized by reductions in the total adenine nucleotide pool, phosphocreatine and glycogen in the slow oxidative soleus, fast oxidative-glycolytic plantaris and the fast glycolytic white gastrocnemius. Compared to ischaemia, the total adenine nucleotide pool was higher (by 7.2-13.3 mumol g-1 dry wt) and the glycogen content lower (by 10.0-16.6 mumol g-1 dry wt) in skeletal muscle exposed to hypoxic perfusion without glucose. The ability of hypoxic perfusion to attenuate TAN degradation and augment glycogenolysis can be attributed to metabolic by-product removal. By limiting muscle lactate and PCO2 accumulation, hypoxic perfusion without glucose attenuates cellular acidification; this could in turn limit AMP deaminase activation and glycogen phosphorylase inhibition. We conclude that the ischaemia-induced alterations in adenine nucleotide and glycogen metabolism arise in response to the elimination of exogenous substrates and to the accumulation of metabolic by-products.

Diadenosine polyphosphates inhibit adenosine kinase activity but decrease levels of endogenous adenosine in rat brain.

Findings in peripheral tissues that diadenosine polyphosphates (Ap n As) activate 5′-nucleotidase activity and inhibit adenosine kinase activity in vitro led us to test the hypothesis that Ap n As and analogues thereof, through such actions on purine enzymes, increase brain levels of endogenous adenosine in vivo. Accordingly, we tested Ap n As for their effects on the in vitro activities of adenosine kinase, adenosine deaminase, AMP deaminase and 5′-nucleotidase and, following unilateral microinjections in rat striatum, on in vivo levels of endogenous adenosine. Adenosine kinase activity was not affected significantly by 5′,5‴-P 1,P 2-diadenosine pyrophosphate (Ap 2A) or by 5′,5‴-P 1,P 3-diadenosine triphosphate (Ap 3A), but was inhibited by 5′,5‴-P 1,P 4-diadenosine tetraphosphate (Ap 4A), 5′,5‴-P 1,P 5-diadenosine pentaphosphate (Ap 5A) and 5′,5‴-P 1,P 6-diadenosine hexaphosphate (Ap 6A); apparent IC 50 values were 5.0, 3.3 and 500 μM, respectively. Inhibition of adenosine kinase activity by Ap 4A and the four metabolically stable analogues of Ap 4A tested was uncompetitive. Following unilateral intrastriatal injections, adenosine levels, relative to uninjected contralateral striatum, were decreased significantly ( P<0.05) by 48% with Ap 4A and by 37% with AppCH 2ppA, a metabolically stable analogue of Ap 4A. Striatal levels of adenosine were not affected significantly by Ap 5A or Ap 6A. Cytosolic, but not particulate 5′-nucleotidase activity was inhibited and AMP deaminase activity was increased by some Ap n As. Although adenosine kinase inhibitors increase levels of endogenous adenosine and we showed here that Ap n As were potent inhibitors of this enzyme, these particular actions of Ap n As were not consistent with their effects on levels of endogenous adenosine.

Adenosine-5'-phosphate deaminase. A novel herbicide target.

The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool.

Effect of endurance training on ammonia and amino acid metabolism in humans

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.

SKELETAL MUSCLE CREATINE KINASE DEFICIENCY (MCK-D) INDUCES HIGH AFFINITY AMP DEAMINASE KINETICS 1307

Depletion of skeletal muscle creatine by feeding a creatine analogue decreases the AMP deaminase (AMPD) capacity and the expression of normal 80 kDa AMPD (Am. J. Physiol. 270:C76-C85, 1996). Kinetic changes in AMPD also occur that decrease the Km at low [AMP]. The muscle creatine kinase knockout mouse (PNAS USA 91:9091-95, 1994) has also been used to study changes in AMPD related to impaired CK flux. Previous work demonstrated an ≈60% decrease in skeletal muscle AMPD capacity, and 80 kDa AMPD in MCK-D compared to wild type (WT) mice. In this study AMPD Vmax(15 mM AMP) was determined using whole homogenates of mixed fast twitch muscle, and AMPD kinetics was determined over a 0.04-15 mM [AMP] range using the supernatants of muscle homogenates. AMPD capacity was decreased ≈65% in MCK-D compared to WT (Table). AMPD kinetics for WT was a single linear phase over the entire [AMP] range, but was biphasic for MCK-D, revealing a novel, high affinity (low Km) reaction at low [AMP]. This kinetic change increases the relative AMPD activity at low [AMP] compared to high [AMP] (increased V.04 mM/V15 mM) in spite of a lower AMPD Vmax. AMPD is kinetically altered when skeletal muscle has impaired CK flux.

Exercise and recovery in frog muscle: metabolism of PCr, adenine nucleotides, and related compounds.

The effects of exercise (swimming), fatigue, and recovery on the intracellular pH (pHi), energy-rich phosphates, and related metabolites were studied in the gastrocnemius muscle of common frogs (Rana temporaria) at 20 degrees C. Exercise caused a rapid decrease in the content of phosphocreatine (PCr) and a corresponding increase in that of Pi. The ATP level remained virtually constant for 1 min; its precipitous decrease during the following minute was associated with a rise in the contents of inosine 5'-monophosphate (IMP) and NH4+, indicating a marked activation of AMP deaminase. Five minutes of swimming caused severe fatigue, which was correlated with decreases in muscle PCr (-85%), ATP (-42%), and pHi (-0.8 units). Recovery appeared almost complete within 2 h, and the frogs were then induced to swim again. During the initial 10 s of this second exercise, ATP synthesis was as high as in the first exercise, but the rate decreased more rapidly between 10 and 60 s, thus indicating that repeated exercise caused increased metabolic stress. IMP formation in working muscle was not strictly correlated with the pHi or the tissue contents of Pi, AMP and ADP, although from studies in vitro AMP deaminase is known to be modulated by these parameters.

Creatine analogue beta-guanidinopropionic acid alters skeletal muscle AMP deaminase activity.

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.

Activation of AMP deaminase in human erythrocytes by calcium ions.

We examined 5'-AMP aminohydrolase (AMP deaminase, EC 3.5.4.6. AMPD) together with metabolite changes in intact red blood cells from normal human adults under different incubation conditions. Moderate changes occurred in erythrocyte adenylates upon incubation for 1 h at 37 degrees C in the absence of glucose and the concentration of IMP was negligible. Calcium in the incubation medium, at concentrations up to 1.50 mmol l-1 did not influence the nucleotide pattern. Inclusion of the ionophore A 23187 in the incubation medium resulted in a sharp decrease of ATP content at 60 min and increases in AMP and ADP concentrations. A maximally low level of ATP was observed at 60 min of incubation in the presence of both the ionophore and 0.25 mmol l-1 of external calcium. With increasing concentrations of calcium up to 1.50 mmol l-1 in the presence of the ionophore, a significant rise occurred in both IMP and ATP concentrations, whether glucose was present or not, and significant correlations existed between the concentrations of calcium and IMP. The presence of the ionophore together with glucose but without extra calcium in the incubation medium produced the highest concentrations of lactate, indicative of an enhanced glycolytic flux under these conditions. The stimulatory effect of intracellular calcium ions on AMPD activity is discussed.

Developmental changes in purine nucleotide metabolism in cultured rat astroglia

The present study was conducted in order to clarify the role of the glia in brain purine metabolism. This, in connection with the clarification of the etiology of the neurological manifestations associated with some of the inborn errors of purine metabolism in man. Purine nucleotide content, the capacity for de novo and salvage purine synthesis and the activity of several enzymes of purine nucleotide degradation, were assayed in primary cultures of rat astroglia in relation to culture age. The capacity of the intact cells to produce purine nucleotides de novo exhibited a marked decrease with the culture age, but the activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), catalyzing salvage nucleotide synthesis, increased. Aging was also associated with a marked increase in the activity of the degradation enzymes AMP deaminase, purine nucleoside phosphorylase (PNP) and guanine deaminase (guanase). The activity of adenosine deaminase and of AMP-5′-nucleotidase, increased markedly during the first 17 days in culture, but decreased thereafter.The results indicate that purine nucleotide metabolism in the cultured astroglia is changing with aging to allow the cells to maintain their nucleotide pool by reutilization of preformed hypoxanthine, rather than by de-novo production of new purines. Aging is also associated with increased capacity for operation of the adenine nucleotide cycle, contributing to the homeostasis of adenine nucleotides and to the energy charge of the cells. In principle, the age-related alterations in purine metabolism in the astroglia resemble those occurring in the maturating neurons, except for the capacity to produce purines de novo, which exhibited inverse trends in the two tissues. However, in comparison to the neurons, the cultured astroglia possess the capacity for a more intensive metabolism of purine nucleotides.

Divergent N-Terminal Regions in AMP Deaminase and Isoform-Specific Catalytic Properties of the Enzyme

AMP deaminase (AMPD) catalyzes the hydrolytic deamination of AMP to IMP and NH 3. This activity is represented throughout mammalian tissues and cells by at least three isoforms. Human AMPD cDNAs have been cloned and sequenced, leading to predictions that each isoform contains distinct amino ends (N-terminal regions) in contrast to their highly conserved carboxyl-ends (C-terminal regions). Wild type, truncated, and chimeric human ARPD1 (isoform M) and AMPD2 (isoform L) cDNAs were expressed and the resultant activities partially characterized as a means to examine the role of divergent N-terminal regions in these polypeptides (residues 1-262 and 1-258 of isoforms M and L, respectively) on isoform-specific catalytic properties. Similar to activities purified from human tissues, in the presence of monovalent cation, wild type isoform M displayed hyperbolic kinetics in the presence and absence of ATP, whereas wild type isoform L, exhibited allosteric activation in the presence of this nucleotide effector. Expression of both a chimeric M (5′-AMPD1)/L (3′-AMPD2) construct and one in which the N-terminal region of isoform L was deleted produced activities that were also allosterically regulated by ATP. However, no AMPD activity was detectable following expression of either a chimeric L (5′-AMPD2)/M (3′-ARPD1) construct or one in which the N-terminal region of isoform RI had been deleted. The N-terminal region also affected the relative ability of each recombinant AMPD activity to deaminate substrate analogs modified in either the sugar or the phosphate, but not in the purine base, moieties of AMP. These combined data show (i) that isoform M, but not isoform L, absolutely requires its N-terminal region for proper function, (ii) that the C-terminal region of isoform L is responsible for allosteric activation by ATP, (iii) an effect of the N-terminal on substrate-enzyme interaction, a contention that is discussed in context with available information regarding the related purine catabolic activity, adenosine deaminase.

Exercise causes branched-chain oxoacid dehydrogenase dephosphorylation but not AMP deaminase binding.

The responses of AMP deaminase (AMPD) and branched-chain oxoacid dehydrogenase (BCOAD) to moderate (70% maximal O2 consumption for 90 min) followed by intense (90% maximal O2 consumption to exhaustion) cycling exercise were evaluated in the active skeletal muscle of human subjects (n = 8). The exercise conditions invoke different energy demands and ammonia production rates. Active muscle and plasma ammonia concentrations continuously increased throughout moderate exercise in the absence of significant inosine 5-monophosphate accumulation. The free activity of AMPD decreased during moderate exercise (by approximately 25-35%), whereas myosin-bound activity did not change. BCOAD was significantly dephosphorylated (activated) at 5 min and was continuously dephosphorylated during moderate exercise (to a maximum of approximately 21%). Ammonia accumulation rate increased dramatically during the higher intensity exercise accompanied by inosine monophosphate accumulation of approximately 2 mmol/kg dry muscle. The higher intensity exercise caused no further changes in AMPD activity distribution or BCOAD dephosphorylation. Resting muscle percent bound AMPD was notably higher than values previously reported for rat muscle. Increases in percent bound AMPD during exercise were the result of decreases in the sum of free and bound activities and not increases in bound activity. The results of this study do not support a role for myosin binding in the activation of AMPD in human skeletal muscle.