AMP deaminase from the gill of Salmo gairdnerii Richardson: effects of anions, cations and buffers

The AMP deaminase isoenzymes from trout gill were activated by sodium and potassium, sodium being the most efficient. The optimal concentration for activation was 30-50 mM. The enzyme was sensitive to ionic strength, and imidazole was an inhibitor at concentrations higher than 25 mM. A possible regulation of gill AMP deaminase by intracellular imidazole buffers is discussed. AMP deaminase activity was tested in the presence of physiological concentrations of sodium and potassium. When the concentration of one of these cations was varied around its physiological concentration, the enzyme activity was relatively stable, indicating that the intracellular AMP deaminase activity would be insensitive to changes in the concentrations of monovalent cations. The effects of the sodium salts of different inorganic and organic anions were tested. Except chloride and gluconate, all were inhibitors of gill AMP deaminase.     

Regulation of trout gill AMP deaminase by lipid bilayers. Effects of phospholipid composition and temperature

Trout gill AMP deaminase is inhibited by liposomes made of synthetic phosphatidylcholines containing higher saturated fatty acids. A preincubation of 1 hr, at 4 degrees C, was necessary to obtain the maximal effect. At 4 or 25 degrees C, these phospholipids modified essentially the substrate affinity of the enzyme by increasing the Michaelis constant proportionally to the length of the fatty acid chain. At 13 degrees C, the liposomes decreased the Hill coefficient also, thus inducing a negative cooperativity. Natural phosphatidylcholine and phosphatidylserine were without significant effect on gill AMP deaminase while natural sphingomyelin exhibited a similar effect to that shown in the presence of synthetic phosphatidylcholines. These results are discussed in relation to a possible effect of sphingomyelins in vivo.     

Activation of trout gill AMP deaminase by an endogenous proteinase--I. Effects on the regulatory properties of the enzyme

Some regulatory properties of trout gill AMP deaminase were determined in crude extracts, before or after modification of the enzyme by the endogenous proteinase. After proteolysis, the optimal concentrations for activation by sodium and potassium were shifted from 10 to 75 mM, resulting in a large increase of enzyme activity near the physiological potassium concentration. This activation was shown to be the consequence of a much lower sensitivity of AMP deaminase to inhibition by increasing ionic strength. The modified enzyme was also less sensitive to modifications of pH and to inhibition by physiological concentrations of inorganic phosphate. When all these modifications were considered, limited proteolysis of gill AMP deaminase resulted in a 40 times increase of enzyme activity under in vivo conditions.     

Role of AMP deaminase reaction in the control of fructose 1,6-bisphosphatase activity in yeast

The physiological role of the inhibition of AMP deaminase (EC 3.5.4.6) by Pi was analyzed using permeabilized yeast cells. (a) Fructose 1,6-bisphosphatase (EC 3.1.3.11) was inhibited only a little by AMP, which was readily degraded by AMP deaminase under the in situ conditions. (b) The addition of Pi, which showed no direct effect on fructose 1,6-bisphosphatase, effectively enhanced the inhibition of the enzyme by AMP increased through the inhibition of AMP deaminase. (c) Pi activated phosphofructokinase (EC 2.7.1.11) and inhibited AMP deaminase activity. AMP deaminase reaction can act as a control system of fructose 1,6-bisphosphatase activity and gluconeogenesis/glycolysis reaction through the change in the AMP level. Pi may contribute to the stimulation of glycolysis through the inhibition of fructose 1,6-bisphosphatase by the increase in AMP in addition to the direct activation of phosphofructokinase.     

AMP deaminase as a control system of glycolysis in yeast. Mechanism of the inhibition of glycolysis by fatty acid and citrate

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.     

Aspects of purine metabolism in the gill epithelium of rainbow trout, Salmo gairdneri Richardson.

1. Enzymes interconnecting the adenylate pool were present in high concentration. 2. AMP and adenosine were easily deaminated by the corresponding enzymes whose high levels were detected. 3. Adenylate was hydrolyzed either by deamination to yield IMP which was further dephosphorylated to inosine or by dephosphorylation to adenosine followed by deamination to inosine. 4. Incubation of gill extract with [-14C]-AMP in the presence and absence of ATP but with adenosine deaminase inhibitors allowed demonstration that ATP controlled the balance between these pathways. 5. Some biochemical properties of 5'-nucleotidase. AMP deaminase and adenosine deaminase were defined. 6. Purine salvage enzymes were also estimated.     

Quantitative structure activity relationship studies on the activation of yeast AMP deaminase by polyamines

Quantitative structure activity relationship studies on the activation of AMP deaminase by polyamines were carried out. Polyamine enhanced the maximal velocity of AMP deaminase without changing the affinity for the substrate AMP. Activation by polyamines of AMP deaminase can be accounted for by the simple Michaelis-Menten mechanism in the presence of ATP. A close correlation between the structure and activation constants for polyamines suggests that the binding of polyamine to AMP deaminase involves primarily polar interactions.     

Activation of trout gill AMP deaminase by an endogenous proteinase--II. Modification of the properties of the enzyme during starvation, pollution and salinity changes

The relative amount of modified AMP deaminase has been determined by taking advantage of the different effects of monovalent cations on the two enzymatic forms. When trout were subjected to different environmental perturbations (starvation, pollution of the water by a pesticide, transfer to sea water or reverse transfer to fresh water), modified AMP deaminase could be detected in the gill extracts. Depending on the nature of the stress and the period of experimentation, 8 to 100% of the enzyme had been modified by limited proteolysis. As a consequence of the much higher activity of the proteolyzed AMP deaminase form, a 2 to 12 times increase of the intracellular AMP deaminase activity could be expected. At the same time, limited proteolysis will modify the regulatory properties of the enzyme, since it can be estimated that 50 to 100% of the enzyme activity expressed in the cell will be an AMP deaminase form less sensitive to inhibition by inorganic phosphate and ionic strength, and to variations of the intracellular pH. Limited proteolysis will result in increased AMP deaminase activity under conditions of increased energy demand, where the concentration of inorganic phosphate is dramatically increased. The consequence should be stabilization of the adenylate energy charge.     

The role of GTP in the regulation of two forms of AMP deaminase from chicken kidney

1. Two molecular forms of AMP deaminase have been revealed by phosphocellulose column chromatography of the chicken kidney extract. 2. The chromatographic, kinetic and regulatory properties of these two forms were similar to these of two enzyme forms previously found in the chicken liver, lizard liver and in rat small intestine. 3. GTP exerted different effect from MgGTP on the activity and kinetic parameters of both AMP deaminase I and II from chicken kidney.     

The regulatory role of spermine and fatty acid in the interaction of AMP deaminase with phosphofructokinase

The role of fatty acid and polyamine in the interaction of AMP deaminase (EC 3.5.4.6)-ammonium system with glycolysis was investigated using permeabilized yeast cells. (1) The addition of fatty acid inhibited the activity of AMP deaminase in situ, resulting in a decrease in the total adenylate pool depletion, and in the recovery of the adenylate energy charge. (2) The addition of fatty acid resulted in an indirect decrease in the activity of phosphofructokinase (EC 2.7.1.11) through a reduced level of ammonium ion; fatty acid itself did not inhibit phosphofructokinase activity in the presence of excess ammonium ion. (3) Spermine protected AMP deaminase from inhibition by fatty acid: the increased ammonium level enhanced phosphofructokinase activity, glycolytic flux and the recovery of the energy charge. In contrast, alkali metals, which are also activators of AMP deaminase had little effect on the inhibition of the enzyme. The inhibition of glycolysis by fatty acid and its reversal by polyamine can be accounted for by the changes in ammonium ion through the action of AMP deaminase-ammonium system, and the physiological relevance is discussed.     

Ambient ammonia effect on catalytic potential of muscle and gill AMP deaminase in fish

AMP deaminase activity was inhibited in gill and muscle of Sarotherodon mossambicus, subjected to a week-long exposure to sublethal concentration of ambient ammonia. pH-dependent kinetic studies revealed decreased catalytic efficiency of the enzyme due to altered active site density and ionization pattern in the fish tissues at different phases of ammonia exposure.     

Pharmacological inhibition of AMP-deaminase in rat cardiac myocytes

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

Pharmacological Inhibition of AMP-Deaminase in Rat Cardiac Myocytes

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

Adenosine-5-monophosphate catabolism in frog liver

1. AMP catabolism in frog liver extract was found to proceed exclusively through the formation of IMP. Further metabolism of IMP is relatively slow. 2. Among the enzymes involved in AMP catabolism, AMP deaminase is most active and adenosine deaminase and AMP 5'-nucleotidase exhibit only 20 and 10% of AMP deaminase activity respectively.     

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

Chromatography on phosphocellulose column revealed changes in the elution profile of 14 day-old chicken embryo and adult hen skeletal muscle AMP deaminase. In the presence of 5 mM potassium the enzyme from embryo muscle exhibited a sigmoid-shaped plot of the reaction rate versus substrate concentration. The increase of KCl concentration up to 100 mM diminished distinctly sigmoidicity of the plot. Micromolar concentrations of ADP or ATP activated, whereas GTP at the same concentrations inhibited the embryo and hen skeletal muscle AMP deaminase while 5 mM KCl was present in the incubation medium. 100 mM potassium concentration diminished the effect of ADP and ATP but not of GTP. Palmitoyl-CoA inhibited strongly the embryo skeletal muscle adenylate deaminase but had no effect on the activity of the hen enzyme. Alanine inhibited only the adult hen enzyme. The embryo and hen AMP deaminase differed also in the specificity to adenylate analogues and exhibited a different dAMP/AMP ratio. The data presented indicate that kinetic and regulatory properties of the two developmental forms of AMP deaminase are different.     

Developmental changes of chicken liver AMP deaminase.

The AMP deaminase activity measured in crude chicken liver extract did not change significantly during development. The livers of 10- and 14-day chick embryos, 1-day, 5-, 10- and 16-week-old chickens and adult hens were examined for the existence of multiple forms of AMP deaminase. Phosphocellulose column chromatography revealed the existence of two peaks of enzyme activity in the liver of 10- and 16-week-old chickens and adult hens. Kinetic studies with the preparations of AMP deaminase revealed sigmoid-shaped substrate-saturation curves at all developmental stages and hyperbolic-shaped saturation curves for the enzyme form appearing in 10-week-old chickens. All AMP deaminases investigated were susceptible to activation by ATP and inhibition by Pi. Kinetic and regulatory properties as well as pH optima of all the enzyme preparations tested indicate that AMP deaminase isolated from the embryos and from 1-day-old chicks was similar to the form I isolated from adult hens and differed significantly from the form II of this enzyme.     

Activation of trout gill AMP deaminase by an endogenous proteinase--III. Comparative studies on the gill AMP deaminase from different fresh water and sea water teleosts

The effects of monovalent cations and inorganic phosphate, on gill AMP deaminase, were compared in different fresh water and sea water stenohaline and euryhaline Teleosts. Generally, sea water species displayed a lower sensitivity to these effectors than fresh water species. During salinity changes, the sensitivity of gill AMP deaminase to cations and phosphate were modified proportionally to the tolerance of a given species to variations of environmental salinity. In particular, these parameters were modified in the weak euryhaline, Salmo gairdneri, but not in the real euryhaline, Anguilla anguilla. In sea water adapted trout, the appearance of a modified AMP deaminase form, with similar properties to that found in sea water species, is suggested. When compared with the conclusions from the preceeding papers [Raffin (1986) Comp. Biochem. Physiol. 85B, 157-162; 85B, 163-171], the results suggest that modification of gill AMP deaminase by limited proteolysis should be a rather general adaptation mechanism to stress.     

腺苷酸脱氨酶在乳酸克鲁维酵母中构建表达

【目的】实现鼠灰链霉菌来源经密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母(Kluyveromyces lactis GG799)中组成型表达。【方法】以鼠灰链霉菌(Streptomyces murinus)来源的腺苷酸脱氨酶(AMP)基因经密码子优化后作为模板,设计特异性引物,PCR扩增AMP脱氨酶基因opt-AMPD,以p KLAC1为载体构建重组表达质粒p KLAC1-opt-AMPD,经Sac II线性化后电转化法转入K.lactis GG799,筛选得到重组菌株,测定酶活,经His TrapTM HP纯化后得到AMP脱氨酶,并优化重组菌的发酵培养基。【结果】对AMP脱氨酶基因进行了密码子优化后,构建了重组K.lactis GG799/p KLAC1-opt-AMPD,实现组成型表达,密码子优化后AMP脱氨酶酶活提高到586±50 U/m L。SDS-PAGE结果显示,纯化后的AMP脱氨酶为单一条带,蛋白大小约为60 k D。优化的发酵培养基为(g/L):葡萄糖40、蛋白胨20、酵母粉15、Na Cl 8、KCl 10、Mg SO4 2,30°C、200 r/min发酵120 h,酶活达到2 100±60 U/m L。【结论】实现了密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母GG799内的组成型表达,为实现腺苷酸脱氨酶的重组高效表达和发酵生产进行了有益探索。     

AMP deaminase from yeast. Role in AMP degradation, large scale purification, and properties of the native and proteolyzed enzyme

Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools. This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4. Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734-8743). The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts. Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region. Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000. The unproteolyzed enzyme is highly unstable during purification. Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal. In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics. ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM. Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively. The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar. Thus, the N-terminal region is not required for catalysis or allosteric activation. AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP. The inhibition constants for these inhibitors decrease in the presence of ATP. ATP, therefore, tightens the binding of GTP, PO4, and AMP. The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism. Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators.     

Purification and properties of trout gill AMP deaminase

Summary The AMP deaminase has been purified 450–500 fold from 20,000 g supernatants from trout gill. The procedure comprised cellulose phosphate and DEAE-cellulose chromatography. The gill appeared to contain different isoenzymes as indicated by different chromatographic behaviour on cellulose phosphate and different heat stabilities. The two major isoenzymes were compared with respect to their pH optima and the effect of temperature, ATP and inorganic phosphate. The pH optimum is about pH 6.7 at low substrate concentration. A second optimum is found in phosphate buffer. The substrate saturation curve is hyperbolic, even in the absence of KCl or ATP. ATP is an activator of the enzyme in the absence of KCl, but is without effect in the presence of monovalent cations. Among the monovalent cations tested, Na⁺ is the most potent activator followed by K⁺ and NH ₄ ⁺ . Inorganic phosphate is an inhibitor of gill AMP deaminase increasing the affinity for its substrate but having no effect on the maximal velocity or the Hill coefficient. The inhibition by phosphate is partially reversed by ATP. ADP and GTP are competitive inhibitors of the enzyme. In addition, the enzyme showed negative cooperativity in the presence of ATP or GTP.