AMP-deaminase from normal and cirrhotic human liver: A comparative study

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.     

AMP-deaminase from human term placenta

AMP-deaminase from human term placenta was chromatographed on a phosphocellulose column and physico-chemical and immunological properties of the purified enzyme were investigated. At physiological pH7.0, in the absence of regulatory ligands (control conditions) studied AMP-deaminase manifested sigmoid-shaped substrate saturation kinetics, with half-saturation parameter (S_0.5) value of about 7 mM. Addition of important allosteric effectors (ATP, ADP or orthophosphate) modified kinetic properties of studied AMP-deaminase, influencing mainly the value of S_0.5 parameter. Micromolar concentrations of stearylo-CoA inhibited potently the enzyme making it no longer sensitive towards 1 mM ATP-induced activation. SDS-PAGE electrophoresis of the purified enzyme revealed presence of 68 kDa protein fragment, reacting with anti-(human) liver AMP-deaminase antibodies. Experimental results presented indicate that liver type of AMP-deaminase is an enzyme form present in human term placenta.     

Comparative studies on heart and skeletal muscle AMP-deaminase from rainbow trout (Salmo gairdneri).

1. AMP-deaminases from fish heart and skeletal muscle have been isolated, and their kinetic and regulatory properties compared. 2. The results obtained indicate that the enzyme variants present in fish heart and skeletal muscle, in contrast to their mammalian counterparts, show very similar chromatographic, kinetic and regulatory characteristics. 3. The above may reflect evolutionary programmed differences in AMP-deaminase gene(s) organization.     

Regulatory properties of 14 day embryo and adult hen heart AMP-deaminase

Chromatography on phosphocellulose column revealed changes in the elution profile of chicken heart AMP-deaminase during ontogenesis. The extracts from the heart of adult hen and 14 day-old embryo displayed a single peak of the enzyme activity at a slightly different elution volume, whereas in the heart extract of 1 day-old chicken two molecular forms of adenylate deaminase have been eluted. The kinetic and regulatory properties of the purified adult hen heart AMP-deaminase were studied and compared with those of the corresponding enzyme from 14 day-old embryo heart. Both enzymes exhibited a slightly sigmoid-shaped plot of the reaction rate versus substrate concentration, which shifted to hyperbolic form when ATP or ADP were added into the incubation medium. The enzymes were strongly activated by ATP, less efficiently by ADP and the activatory effect was enhanced at low substrate concentration. Orthophosphate inhibited both enzymes but this inhibition was more potent for the embryo heart enzyme. Palmitoyl-CoA inhibited adult hen but not the embryo heart AMP-deaminase. The data presented indicate that the differences also in the regulatory properties of the molecular forms studied do exist and correspond with the ontogenetic differences observed previously (Kaletha and Skladanowski (1981) Experientia 37, 232-234) concerning the effect of temperature on the chicken heart adenylate deaminase.     

Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP-deaminase.

The effect of oxidative stress on human red blood cell AMP-deaminase activity was studied by incubating either fresh erythrocytes or hemolysates with H(2)O(2) (0.5, 1, 2, 4, 6, 8, and 10 mm) or NaNO(2) (1, 5, 10, 20, and 50 mm), for 15 min at 37 degrees C. AMP-deaminase tremendously increased by increasing H(2)O(2) or NaNO(2) at up to 4 and 20 mm, respectively (maximal effect for both oxidants was 9.5 and 6.5 times higher enzymatic activity than control erythrocytes or hemolysates, respectively). The incubation of hemolysates with iodoacetate (5-100 mm), N-ethylmaleimide (0.1-10 mm), or p-hydroxymercuribenzoate (0.1-5 mm) mimicked the effect of oxidative stress on AMP-deaminase, indicating that sulfhydryl group modification is involved in the enzyme activation. In comparison with control hemolysates, changes of the kinetic properties of AMP-deaminase (decrease of AMP concentration necessary for half-maximal activation, increase of V(max), modification of the curve shape of V(o) versus [S], Hill plots, and coefficients) were recorded with 4 mm H(2)O(2)- and 1 mm N-ethylmaleimide-treated hemolysates. Data obtained using 90% purified enzyme, incubated with Fenton reagents (Fe(2+) + H(2)O(2)) or -SH-modifying compounds, demonstrated that (i) reactive oxygen species are directly responsible for AMP-deaminase activation; (ii) this phenomenon occurs through sulfhydryl group modification; and (iii) the activation does not involve the loss of the tetrameric protein structure. Results of experiments conducted with glucose-6-phosphate dehydrogenase-deficient erythrocytes, challenged with increasing doses of the anti-malarial drug quinine hydrochloride and showing dramatic AMP-deaminase activation, suggest relevant physiopathological implications of this enzymatic activation in conditions of increased oxidative stress. To the best of our knowledge, this is the first example of an enzyme, fundamental for the maintenance of the correct red blood cell energy metabolism, that is activated (rather than inhibited) by the interaction with reactive oxygen species.     

AMP-deaminase activity in denervation atrophy of the amphibian skeletal muscle

The substrate (AMP) and co-factor (ATP)-dependent kinetic parameters of AMP-deaminase have been studied in the contralateral and denervated gastrocnemius muscles of frog, Rana hexadactyla. An increasing in apparent affinity (Km) and maximal velocity (V) were found with denervated muscle enzyme as compared to the contralateral muscle enzyme. The activation energy (delta E) values were found to be decreased on denervation suggesting increased catalytic efficiency of denervated muscle enzyme.     

Inhibition by alanine of AMP-deaminase from rabbit skeletal muscle.

1. Alanine inhibits rabbit muscle AMP-deaminase while aspartate, histidine and glutamate are ineffective. 2. The degree and type of inhibition of AMP-deaminase by alanine depend on pH; at pH 6.5 alanine behaves like an allosteric effector exerting a negative heterotropic effect. At pH 7.0 the inhibition is non-competitive, Ki being as high as 19 mM. 3. The probable significance of the effect of alanine on AMP-deaminase in muscle metabolism is discussed.     

Human liver AMP-deaminase – oligomeric forms of the enzyme

AMP-deaminase (EC 3.5.4.6) is a key enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in the liver. Mechanisms regulating activity of the enzyme are not completely elucidated, till now. In this paper experimental data indicating on the potential regulatory significance of changes in oligomeric structure of the enzyme are presented. SDS-PAG electrophoresis of human liver AMP-deaminase revealed the presence of three enzyme fragments. Only largest of them (the protein fragments weighing 68 kDa) reacted immunologically with monoclonal anti- (human liver) AMP-deaminase antibodies. At physiological pH 7.0, in the absence of regulatory ligands, reaction catalysed by human liver AMP-deaminase was strongly dependent on enzyme concentration used, with half-saturation constant (S_0.5) values increasing significantly with the degree of enzyme dilution. Preincubation with activated long-chain fatty acids – substances promoting dissociation of oligomeric enzymes, inhibited the activity of AMP-deaminase studied nearly completely. Gel filtration on Sepharose CL-6B column demonstrated existence of at least three active oligomeric forms of human liver AMP-deaminase. We postulate that oligomeric structure of the enzyme is a factor determining regulatory profile of AMP-deaminase studied.     

AMP-deaminase activity of circulating leukocytes in the human

It is shown that the AMP-deaminase activity in leucocytes of the human peripheric blood in contrast with the enzyme from erythrocytes manifests its activity only if it is isolated in the presence of K+ or Na+ ions. Pi and GTP being inhibitors of the enzyme in different tissues including erythrocytes do not alter the AMP-deaminase activity in leucocytes. 3,3',5-triiodothyracetic acid markedly decreasing the AMP-deaminase activity of leucocytes does not affect the enzyme activity in the hemolyzate of erythrocytes. The results obtained have shown that the AMP-deaminase activity in leucocytes of the human peripheric blood possesses some regulatory properties differing from those of the enzyme in erythrocytes.     

Phosphorylation of the skeletal muscle AMP-deaminase by protein kinase C

Protein kinase C catalyzes phosphorylation of the rat skeletal muscle AMP-deaminase in the presence of calcium ions and phosphatidylserine. At the same time, the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate AMP-deaminase. Ca2+, phosphatidylserine-dependent phosphorylation decreases three-fold (from 0.6 to 0.2 mM) the Km value and does not affect Vmax. Protein kinase C-induced phosphorylation of AMP-deaminase, besides ADP-ribosylation, is suggested to be involved in regulating the AMP-deaminase activity in vivo.     

OPERATION OF THE PURINE NUCLEOTIDE CYCLE IN ANIMAL TISSUES

1. The operation of the purine nucleotide cycle, consisting of the enzymes adenylate deaminase (E.C. 3.5.4.6), adenylosuccinate synthetase (E.C. 6.3.4.4) and adenylosuccinate lyase (E.C. 4.3.2.2), has been reviewed with reference to its metabolic function in animal tissues.
2. Abundant evidence, both from in vitro and in vivo studies, suggests that the purine nucleotide cycle serves to stabilize the adenylate 'energy charge' (or 'phosphorylation potential') in the cytoplasm of vertebrate cells during a temporary imbalance between ATP-consumption and ATP-production. This stabilization, however, is absent or much less efficient in tissues of invertebrates.
3. The hypothesis that AMP-deaminase is involved in the regulation of glycolysis is not supported by recent work. In a variety of cell types, including skeletal muscle and blood platelets, blocking of AMP-deaminase activity (due to a genetic defect or to pharmacological inhibition) is without effect on the glycolytic rate. Detailed kinetic and histochemical analysis of energy metabolism shows lack of correlation between AMP-deaminase activity and glycolysis in skeletal muscle during exercise.
4. The purine nucleotide cycle appears to control the level of citric acid cycle intermediates in skeletal muscle. Pharmacological inhibition of adenylosuccinate lyase or adenylosuccinate synthetase leads to a reduced availability of four-carbon 'sparker' molecules to the Krebs cycle with a concomitant impairment of aerobic energy production during muscular work.
5. The cycle appears to be a major pathway for amino acid deamination in skeletal muscle and brain of vertebrates, but not in kidney or liver.     

Erythrocyte AMP-deaminase: an investigation of the increase in activity during chick maturation.

1. AMP-deaminase activity in erythrocytes increases gradually during chick (Gallus domesticus) maturation, reaching the adult level of enzymatic activity at about 16 weeks after hatching. 2. Adenosine deaminase activity increases approximately two-fold during this period. 3. Substrate specificity and immunoinhibition studies indicate that erythrocytes from adult chickens and newly-hatched chicks contain the same AMP-deaminase isozyme. 4. Comparison of temporal changes in RBC AMP-deaminase with those previously described for this enzyme in muscle and brain suggests that the level of this enzyme is regulated differently in these tissues.     

Role of adenylate kinase, AMP deaminase and 5'-nucleotidase in the metabolism of adenylic nucleotides

The activities of adenylate kinase, AMP-deaminase and 5'-nucleotidase in various tissues of the rat were studied. The activity of the forward adenylate kinase reaction (ATP + AMP----2 ADP) against the back one (2 ADP----ATP + AMP) was predominant. The liver was shown to contain two, while the blood serum--three adenylate kinase isoenzymes. In the skeletal muscles, the catabolism of adenylic acid involving AMP-deaminase and 5'-nucleotidase predominantly occurred via deamination, in the liver--via dephosphorylation, while in the leucocytes, erythrocytes and blood serum the activity of these processes was essentially the same. In vitro, ATP enhanced the activity of AMP-deaminase in the liver, leucocytes and erythrocytes and decreased it in the blood serum. Under effects of ATP, the activity of 5'-nucleotidase in the leucocytes and blood serum was markedly elevated, that in the liver and erythrocytes was unaffected.     

Degradation of adenine nucleotides in the erythrocytes of patients with chronic renal failure

In chronic renal failure AMP-deaminase operates in the erythrocytes at a much higher velocity than in healthy subjects, with a simultaneous shift from the AMP-adenine-inosine-hypoxanthine pathway (55% and 19%, respectively) to the pathway initiated by AMP-deaminase (45% and 81%, respectively).     

AMP-deaminase in elasmobranch fish: a comparative histochemical and enzymatic study

AMP-deaminase activity was measured in white muscle from a wide range of fish, including one cyclostome, 13 chondrosteans, and one teleost to elucidate the pattern of the AMP-deaminase activity in white muscle of fish. Compared to a mammalian (rat) muscle extract, low enzyme activities are found in the cyclostome and two elasmobranchs from two families (Scyliorhinidae, Hexanchidae). In contrast, higher AMP-deaminase activities, similar to mammals, are expressed in Squalidae, all families of skates, Chimaeridae and in the teleostean fish. We then compared AMP-deaminase activities in red and white muscles from two representative elasmobranch fish, the dogfish (Scyliorhinus canicula) and the thornback ray (Raja clavata). The fibre type composition and distribution of the locomotory musculature were determined in these two elasmobranchs to establish a relationship between the morphology, the type of fibres of the locomotion-implicated muscles and the AMP-deaminase activity. Experimental data are discussed with respect to the layout of fibres in the myotome. In both species, three fibre types were identified. In the two fish myotomes, most of the axial muscles are white fibres while red fibres constitute a thin sheet. Some differences were observed between the two species in the distribution of intermediate fibres: in dogfish, these are located between the red and white fibres; in thornback ray, some are dispersed within the white fibre region, while others form an intermediary layer like in dogfish. These results suggest that in the course of evolution, an amplification of the AMP-deaminase activity in muscle was coupled with increase of complexity of the muscular structure.     

Subunit structure of AMP-deaminase from skeletal muscles

AMP-deaminase was purified from skeletal muscle of rat by the affinity chromatography on phosphocellulose and gel-filtration on Sephadex G-200. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) has shown three protein bands on each step of purification. One of them corresponds to the subunit of tetrameric AMP-deaminase molecule with molecular weight of 76 kDa and two others--to the protein subunit with molecular weight of 42 and 33 kDa. Repeated SDS-PAGE of the main subunit band has revealed again all these protein bands. The data obtained indicate that AMP-deaminase subunit of 76 kDa is able to dissociate on two polypeptide chains with similar values of molecular weights in the presence of SDS.     

Regulatory effect of pig heart phospholipids on heart muscle AMP-deaminase

AMP-deaminase purified from pig heart has been found to be activated by liposomes prepared from phospholipids extracted from pig heart mitochondria, microsomes and cytoplasm, as well as by intact microsomes. The activation by phospholipids occurred only in the presence of ATP and after the enzyme had been preincubated with liposomes for 30 min. Liposomes prepared from cardiolipin displayed an inhibitory effect on pig heart AMP-deaminase.     

Regulation of avian erythrocyte AMP-deaminase.

1. Kinetic data for avian erythrocyte AMP-deaminase in lysate supernatants and 2000-fold purified enzyme were consistent with an allosteric model having four binding sites for substrate. 2. Relative to the purified enzyme, AMP-deaminase in lysate supernatants exhibited a greater S0.5 and enhanced sensitivity toward phytic acid, but was far less sensitive toward potassium ion. 3. In the absence of potassium chloride, the enzymatic activity in lysates exhibited hysteresis at subsaturating 5'-AMP. This response was modified reversibly by allosteric ligands. 4. It is concluded that the characteristics of avian RBC AMP-deaminase, as expressed in lysates, may reflect important intermolecular interactions and better represent the regulatory properties of this enzyme in erythrocytes.     

Hen heart AMP-deaminase--the combined effect of ATP, ADP and orthophosphate on the enzyme activity

Interpretation of the kinetic data in terms of concerted transition theory indicated that in the presence of 100 mM potassium chloride hen heart AMP-deaminase may be active as a dimer. The presence of ATP, but not of the ADP in the incubation medium shifts completely the allosteric equilibrium towards the active, accessible to the substrate form of the enzyme. In the joint presence of main enzyme effectors (ATP, ADP and orthophosphate) added to the incubation medium at physiological concentrations, the plot of the reaction rate versus substrate concentration manifested hyperbolic dependence and the value of half-saturation constant (K0.5) did not differ from the value of this parameter obtained for ATP(alone)-activated enzyme.