AMP-deaminase from hen stomach smooth muscle--physico-chemical properties of the enzyme

AMP-deaminase from hen stomach smooth muscle was isolated and physico-chemical properties of the purified enzyme were investigated. The enzyme had an activity optimum at pH 6.5, and poorly deaminated the substrate analogues tested. At optimum pH (6.5), in the absence of regulatory ligands (control conditions), the enzyme manifested hyperbolic substrate-saturation kinetics with half-saturation constant (S(0.5)) of about 4.5 mM. Additions of adenine nucleotide effectors (ATP, ADP) activated the enzyme strongly at all the concentrations tested, diminishing significantly the value of S(0.5) constant. In contrast, the regulatory effect of orthophosphate was variable, and depended on the orthophosphate concentration used. The molecular mass of the enzyme subunit determined in SDS/PAG electrophoresis was about of 37kDa. The obtained results suggest that in different types of hen muscle, similarly as in humans and rats, expression of AMP-deaminase is under the control of independent genes.     

Purification and properties of AMP-deaminase from human uterine smooth muscle. Regulation by adenylate energy charge and activated fatty acids

At pH 7.0 and physiological concentrations of the main regulatory ligands (ATP, ADP, orthophosphate), human uterine muscle AMP-deaminase follows a hyperbolic type of saturation kinetics with S0.5 parameter value about 2 mM. The enzyme is regulated by adenylate energy charge (AEC) variations, being the most active at the AEC value 0.5-0.6 or 0.5-0.7, depending on the size of the total adenine nucleotide pool. Long-chain acyl-CoA strongly inhibit activity of the enzyme, influencing mainly the maximum velocity of the reaction.     

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.     

Purification and properties of AMP-deaminase from human kidney.

AMP-deaminase from human kidney (cortex and medulla) was purified and the physicochemical properties were characterized. The enzyme from both portions of the kidney exhibited identical kinetics and regulatory properties. At optimal pH (6.6), the AMP-deaminase studied exhibited a distinctly sigmoidal substrate saturation kinetics, with the half-saturation parameter (S0.5) as high as 10 mM. ATP at 1 mM strongly activated the enzyme, decreasing S0.5 nearly 10-fold. The activating effect of ADP was less strong. Orthophosphate inhibited the enzyme, but the inhibition observed was weak (Ki approximately 16 mM) and had a pure competitive character. At pH 7.2, physiological for the kidney cortex, orthophosphate inhibition became even weaker and became partially competitive. Variations in the adenylate energy charge had potent effects on the activity of AMP-deaminase, depending on the size of the total adenine nucleotide pool examined. The results of gel filtration and SDS-PAGE indicated that human kidney AMP-deaminase is an oligomeric enzyme composed of four, probably identical, subunits weighing about 37 kDa each.     

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.     

Regulation of AMP-deaminase activity from white muscle of common carp Cyprinus carpio

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

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.     

Regulatory properties of pigeon heart muscle AMP deaminase

The kinetic and regulatory properties of purified pigeon heart muscle AMP deaminase were investigated. In the presence of 100 mM potassium chloride, the enzyme exhibited a slightly sigmoidal type of kinetics. Addition of ATP to the incubation medium changed the reaction rate versus substrate concentration plot into a hyperbolic one, and caused a decrease of the half-saturation constant (S0.5). ADP presence caused the change of both the S0.5 and Vmax parameters, exerting either an activating or inhibitory effect, depending upon the substrate concentration. Orthophosphate inhibited the enzyme at all substrate concentrations, increasing the value of the S0.5 parameter. In the presence of ATP, ADP and orthophosphate, added to the incubation medium at approximately physiological concentrations, pigeon heart AMP deaminase still seems to preserve its activated form. Active long chain fatty acids clearly inhibited enzyme activity even at micromolar concentrations. Interpretation of the kinetic data in terms of the allosteric theory of Monod et al. (1965, J. Mol. Biol. 12, 88-118) indicates that heart muscle AMP deaminase may operate as a functionally active dimer.     

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.     

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.     

Inactivation of AMP-deaminase from white muscle of Cyprinus carpio in the systems with free radical oxidation

The purification and in vitro inactivation of AMP-deaminase from white muscle of carp Cyprinus carpio were conducted in the Fe2+/H2O2 and Fe2+/ascorbate oxidation systems. The enzyme activity decreases by 50% within 30 minutes of incubation in the presence of 100 microM of hydrogen peroxide and 5 microM of ferrous sulfate. Inactivation depended on incubation time and concentrations of FeSO4 and H2O2. In the system Fe2+/ascorbate the enzyme activity decreased by 50% at concentration of ascorbate 1 mM and 5 ferrous sulfate microM. Sodium nitrite did not affect the activity. S(0.5) and n(H) of both native and partially inactivated enzymes were virtually the same, while maximal activity of the inactivated enzyme was 2-3-fold lower than that of the native one.     

AMP-deaminase from human skeletal muscle. Subunit structure,amino-acid composition and metal content of the homogenous enzyme

• 1.1. Homogenous human skeletal muscle AMP-deaminase was obtained by chromatography on phosphocellulose.
• 2.2. Native enzyme molecular weight was 290,000, while a value of 71,000 was found for the subunit molecular weight.
• 3.3. No distinct differences were found in amino-acid composition of human skeletal muscle AMP-deaminase as compared with other vertebrate enzymes.
• 4.4. Human muscle AMP-deaminase contains about 2g-atom of zinc per 280,000; considerable amounts of calcium and magnesium were also found.

     

Polynucleotide kinase from rat-liver nuclei. Purification and properties.

A polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid in the presence of adenosine triphosphate, has been purified 260-fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei. The purified enzyme has a pH optimum of 5.5. The enzyme is reversible inhibited by p-chloromercuribenzoate. The S0.5 value (ligand concentration required for a half-maximal activity) for ATP is 2.5 muM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkable inhibits the activity with I0.5 value (ligand concentration required for a half-maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM. An apparent molecular weight of the purified enzyme is estimated to be 8 X 10(4) by gel filtration on a column of Sephadex G-150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.     

Modification by liposomes of the adenosine triphosphate-activating effect on adenylate deaminase from pig heart.

Adenylate deaminase (AMP deaminase, EC 3.5.4.6) of a high substrate specificity was purified from pig heart by chromatography on cellulose phosphate. The enzyme shows a co-operative binding of AMP [h (Hill coefficient) 2.35, with SO.5 (half-saturating substrate concentration) 5mM]. ATP and ADP act as positive effectors, lowering h to 1.55 and SO.5 to 1 mM. The addition of liposomes (phospholipid bilayers) to ATP-activated or ADP-activated enzyme causes a further shift of the h value to 1.04 and SO.5 to 0.5 mM. For ATP-activated enzyme the addition of liposomes increases Vmax. by about 100%, and for ADP-activated enzyme by 50%. Liposomes have no effect on the kinetics of AMP deaminase in the absence of ATP and ADP, and neither do they influence the inhibitory effect of orthophosphate on heart muscle AMP deaminase. Metabolic implications of these findings are discussed.     

Isolation and regulatory mechanisms of smooth muscle AMP-deaminase.

AMP-deaminase from cow uterine smooth muscle has been purified. The enzyme activity is regulated by the two cooperating mechanisms: allosteric and dissociation--association.     

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.     

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.     

Adenylate deaminase from rat muscle. Regulation by purine nucleotides and orthophosphate in the presence of 150 mM KCl.

Adenylate deaminase from rat skeletal muscle has been studied with the objective of understanding how the activity of the enzyme is regulated in vivo. ATP and GTP inhibit the enzyme at low concentrations in the presence of 150 mM KCl. The ATP inhibition is reversed as the ATP concentration is raised to physiological levels. The GTP inhibition is reversed as the GTP concentration is raised to unphysiologically high levels. In the presence of physiological concentrations of ATP, the GTP inhibition is also greatly diminished, but inhibition by orthophosphate remains strong. The apparent affinities of the enzyme for GTP, ATP, and orthophosphate are reduced as the pH is decreased from 7.0 to 6.2. ADP also reduces the apparent affinities of the enzyme for the inhibitors. The regulatory effects of GTP, ATP, and ADP are produced primarily by their unchelated forms. Comparison of the kinetic behavior of the enzyme in vitro with metabolite concentrations in vivo indicates that the major variables that regulate the activity of adenylate deaminase of muscle in vivo are the concentrations of AMP, ADP, orthophosphate, and H+.     

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.     

Effect of spermine on activity of purified Ca2+, Mg2+-ATPase from plasma membranes of myometrium cells

Effect of endogenous polyamine spermine, a relaxant of smooth muscle, on the activity of myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase was studied. It was observed a tendency to activation of enzyme at the spermine concentrations 0.1-0.5 mM, the increase of the polyamine concentrations up to 10 mM inhibited. ATPase by 80% (I50 = 5.5 +/- 0.3 mM). Spermine inhibited enzyme decreasing its turnover rate and affinity for Ca2+. The ATPase affinity for Mg2+ increased in the presence of spermine. It was revealed, that the inhibitory effect of spermine is changed by the stimulatory effect under the increase of Ca2+ concentration (up to 2.6 microM), that correlates with the relaxing effect of this polyamine on the smooth muscle.