Regulatory properties of rat heart AMP deaminase.

The kinetic and regulatory properties of purified rat heart AMP deaminase were investigated. In the presence of 100 mM KCl, the enzyme exhibited a slightly sigmoid-shaped plot of reaction rate, vs. substrate concentration, which shifted to a more hyperbolic form when ATP, ADP or GTP were added. ATP was the most potent activator of the enzyme, whereas GTP at low (less than 0.25 mM) concentrations increased the enzyme activity. The activation effect was negligible at higher concentrations of GTP. The calculated value of K0.5 of approx. 3 mM for unactivated enzyme decrased to approx. 0.6 mM and 1.1 mM when 0.5 mM ATP or 1.5 mM ADP were present in the incubation mixture, respectively. The theoretical model (Monod, J., Wyman, J. and Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118) gave a partial explanation of these results.     

Molecular forms of pigeon skeletal muscle AMP deaminase

Phosphocellulose chromatography of pigeon leg muscle extract revealed the existence of two well-separated forms of AMP deaminase. This was in contrast to the pigeon breast muscle extract, which yielded only one form. The two leg muscle enzyme isoforms manifested similar kinetic and regulatory properties. They were activated by very low concentration of potassium ions and demonstrated similar patterns of pH and effector dependence. At pH 6.5, as well as at other pH values tested. ADP and ATP slightly stimulated, whereas GTP and orthophosphate inhibited the two molecular forms of pigeons leg muscle enzyme. Surprisingly, the molecular form of AMP deaminase present in pigeon breast muscle was inhibited by ATP at all pH values tested. The kinetic and regulatory properties of the three molecular forms of pigeon skeletal muscle AMP deaminase examined do not resemble those which have been described for pigeon heart muscle enzyme.     

Regulatory properties of AMP deaminase isoenzymes from rabbit red muscle.

We examined the kinetic and regulatory properties of the two isoenzymes of red muscle AMP deaminase, forms A and B, corresponding respectively to the single isoenzymes present in the heart and white skeletal muscle. At the optimal pH value, 6.5, both enzymes show hyperbolic substrate-velocity curves and are inhibited by GTP, inducing sigmoid kinetics. An effect similar to that of GTP is exerted on form B by ATP, whereas form A is almost insensitive to this nucleotide. At pH 7.1 both enzymes follow sigmoid kinetics. ATP enhances the sigmoidicity of the substrate-velocity curve of form B, but it stimulates form A, reverting sigmoidal to hyperbolic kinetics shown by the enzyme at optimal pH. At pH 7.1, form A is also less sensitive to the inhibitory action of Pi and GTP. These results suggest that, owing to the presence of form A, AMP deamination occurs in red muscle also at moderate work intensity. A possible role of this process in counteracting the production of adenosine by 5'-nucleotidase is hypothesized.     

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.     

Molecular forms of human heart muscle AMP deaminase.

Chromatography on phosphocellulose revealed the presence of two, kinetically different forms of human heart AMP deaminase. The main portion of the activity eluted from the column at about 1.1 M KCl, and the enzyme present in this eluate manifested a sigmoidal type of substrate saturation kinetics. The results from gel filtration indicate that human heart AMP deaminase is a tetrameric protein capable of aggregating in more complex active structures.     

Determination of AMP in the rat heart using skeletal muscle AMP deaminase

A new simple enzymatic method for measuring AMP content in freeze-clamped rat heart is presented. The method is based on the ammonia estimation after the deamination of 5'-AMP by muscle 5'-adenylic acid deaminase. The minimum detectable amount of AMP was about 1.5 nmol. The recovery of AMP added to the tissue homogenate was 94%. The variance coefficient evaluated by assaying five samples from one tissue extract was equal to 5%. AMP content of rat heart (0.28 mumol/g wet tissue) is comparable with the values reported by others.     

Interaction of rat muscle AMP deaminase with myosin. II. Modification of the kinetic and regulatory properties of rat muscle AMP deaminase by myosin.

The problems of whether the kinetic and regulatory properties of AMP deaminase were modified by formation of a deaminase-myosin complex were investigated with an enzyme preparation from rat skeletal muscle. Results showed that AMP deaminase was activated by binding to myosin. Myosin-bound AMP deaminase showed a sigmoidal activity curve with respect to AMP concentration in the absence of ATP and ADP, but a hyperbolic curve in their presence. Addition of ATP and ADP doubled the V value, but did not affect the Km value. Myosin-bound AMP deaminase also gave a sigmoidal curve in the presence of alkali metal ions, whereas free AMP deaminase gave a hyperbolic curve. GTP abolished the activating effects of both myosin and ATP.     

Comparison of kinetic and regulatory properties of high S0.5 form of AMP deaminase from chicken and pigeon liver with AMP deaminase from rat and ox liver

1. The high S0.5 form of AMP deaminase from avian liver was shown to display a two times lower S0.5 value than the single mammalian enzyme form. 2. Avian enzymes showed several fold higher affinity to the activator (ATP) but lower affinity to inhibitors (GTP and Pi) than the mammalian AMP deaminases. 3. GTP was shown to exert a biphasic: activating and inhibitory effect on all the enzymes tested, the chicken and pigeon enzymes being activated within a much broader range of effector concentration. 4. In the presence of 3 mM ATP the activity of avian enzymes was not affected by high GTP and Pi concentrations, in contrast to AMP diaminase from rat liver which was strongly inhibited by GTP under the same experimental conditions. 5. The differences of the regulatory properties described are discussed in terms of adjustment of avian liver AMP deaminase to a faster adenylates' catabolism and thus urate synthesis.     

AMP deaminase from equine muscle: purification and determination of regulatory properties.

1. AMP deaminase from thoroughbred horse muscle was purified to apparent homogeneity and its regulatory properties were determined at pH 6.5 and 7.4. 2. The results are discussed in relation to the potential role of muscle AMP deaminase during exercise and the existence of two molecular forms depending on the pH.     

Diadenosine tetraphosphate activates AMP deaminase from rat muscle

Diadenosine tetraphosphate, Ap4A, doubled the activity of AMP deaminase from rat muscle, with an activation constant of 0.005 mM, in the presence of 0.05 mM AMP. The presence of Ap4A appeared to induce Michaelian kinetic behavior. The activation by Ap4A was not dependent on the presence of either MgCl2 or KCl in the reaction mixture. Diguanosine tetraphosphate was inhibitor of the enzyme. Diadenosine and diguanosine triphosphates, adenylosuccinate and xanthosine monophosphate were neither inhibitors nor activators of the reaction.     

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.     

AMP deaminase: Stage-specific isozymes in differentiating chick muscle

The molecular basis of the developmental increase in AMP deaminase activity in chick muscle was investigated with a view toward determining whether isozymes of AMP deaminase exist in embryonic avian muscle and, if so, whether a stage-specific isozyme transition occurs during myogenesis in vivo and in vitro. Under specified conditions, AMP deaminase isozymes in adult chicken brain and muscle may be distinguished on the basis of differences in relative substrate specificities for 5′-dAMP and 5′-AMP (expressed as a ratio of the rates observed with these compounds; i.e., $\text{dAMP}\text{AMP}$ ratios), as well as by differential immunoinactivation by antibody directed against breast muscle AMP deaminase. It was found that the AMP deaminase(s) that is (are) present in 6-day embryos is (are) catalytically and immunologically similar to the enzyme in adult brain. With mixtures of known amounts of adult muscle and brain enzymes, values for the $\text{dAMP}\text{AMP}$ ratio (as well as the fraction of uninactivated AMP deaminase at antibody excess) were proportional to the fraction of muscle isozyme present. Standard curves constructed from these data were used to determine that the fraction of adult muscle-like AMP deaminase in developing muscle, as assessed by $\text{dAMP}\text{AMP}$ ratios (and differential immunoinactivation), on days 6, 8, 10, and 15 were 23 (28), 55 (65), 83 (85), and 93% (96), respectively, Thus, parallel results were obtained for the two techniques, and the isozyme transition is virtually complete by the 15th day of incubation. Primary muscle cultures were used to investigate the isozyme transition of AMP deaminase during myogenesis in vitro. Comparison of the data obtained from primary muscle cultures treated with bromodeoxyuridine, cytosine arabinoside, and fluorodeoxyuridine with data from control cultures showed that biochemical differentiation of AMP deaminase in vitro could be attributed to the muscle cell. Also, the isozyme composition changed from a small percentage of adult muscle-like isozyme at the time of plating, to approximately 100% by the 6th day of culture.     

Interaction of rat muscle AMP deaminase with myosin: I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle

AMP deaminase was completely solubilized from rat skeletal muscle with 50 mM Tris-HCl buffer (pH 7.0) containing KCl at a concentration of 0.3 M or more. The purified enzyme was found to be bound to rat muscle myosin or actomyosin, but not to F-actin at KCl concentrations of less than 0.3 M. Kinetic analysis indicated that 1 mol of AMP deaminase was bound to 3 mol of myosin and that the dissociation constant (K_d) of this binding was 0.06 μM. It was also shown that AMP deaminase from muscle interacted mainly with the light meromyosin portion of the myosin molecule. This finding differs from that of Ashby and coworkers on rabbit muscle AMP deaminase, probably due to a difference in the properties of rat and rabbit muscle AMP deaminase.AMP deaminase isozymes from rat liver, kidney and cardiac muscle did not interact with rat muscle myosin. The physiological significance of this binding of AMP deaminase to myosin is discussed.     

Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle

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).     

Interaction of rat muscle AMP deaminase with myosin. I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle.

AMP deaminase was completely solubilized from rat skeletal muscle with 50 mM Tris-HCl buffer (pH 7.0) containing KCl at a concentration of 0.3 M or more. The purified enzyme was found to be bound to rat muscle myosin or actomyosin, but not to F-actin at KCl concentrations of less than 0.3 M. Kinetic analysis indicated that 1 mol of AMP deaminase was bound to 3 mol of myosin and that the dissociation constant (Kd) of this binding was 0.06 micrometer. It was also shown that AMP deaminase from muscle interacted mainly with the light meromyosin portion of the myosin molecule. This finding differs from that of Ashby and coworkers on rabbit muscle AMP deaminase, probably due to a difference in the properties of rat and rabbit muscle AMP deaminase. AMP deaminase isozymes from rat liver, kidney and cardiac muscle did not interact with rat muscle myosin. The physiological significance of this binding of AMP deaminase to myosin is discussed.