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.     

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 AMP deaminases from rat tissues.

1. Phosphocellulose column chromatography under double gradient conditions (phosphate and KCl) revealed two forms of AMP deaminase in rat heart and brain and a single form in the liver and skeletal muscle. 2. Kinetically all purified AMP deaminases were classified into two categories: those, which elute from the column at lower KCl and Pi concentrations, display low S0.5 value are only moderately affected by MgATP, MgGTP and Pi; and those which elute at higher KCl and Pi concentrations, display high S0.5 values and are strongly regulated by allosteric effectors. 3. Physiological significance of the occurrence of two kinetic forms of AMP deaminase in some tissues is discussed.     

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.     

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.     

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.     

AMP deaminase from baker's yeast. Kinetic and molecular properties.

The kinetic and molecular properties of AMP deaminase [AMP aminohydrolase, EC 3.5.4.6] purified from baker's yeast (saccharomyces cerevisiae) were investigated. The enzyme was activated by ATP and dATP, but inhibited by Pi and GTP in an allosteric manner. Alkali metal ions and alkaline earth metal ions activated the enzyme to various extent. Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates. Kinetic analysis showed that the number of interaction sites for AMP (substrate) and Pi (inhibitor) is two each per enzyme molecule. The molecular weight of the native enzyme was estimated to be 360,000 by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83,000, suggesting that the native enzyme has a tetrameric structure. Baker's yeast AMP deaminase was concluded to consist of two "promoter" units which each consist of two polypeptide chains with identical molecular weight.     

Phorbol esters and cyclic AMP activate AMP deaminase in adult rat cardiac myocytes.

Using rapid deenergization as a probe for adenylate deaminase activity in intact adult rat cardiac myocytes, we have previously established that IMP formation is enhanced by alpha-adrenergic agonists. In the present study, the effect of adrenergic agents on adenylate deaminase was further characterized. Phenylephrine (PE)3 increased IMP production in a dose-dependent fashion with an EC50 of 8 x 10(-7) M. The response to PE was reversed within 10 min by the alpha 1-antagonist, prazosin. Likewise, adenylate deaminase was also activated in ventricular myocytes challenged with phorbol 12-myristate 13-acetate (PMA, EC50 = 5 nM); cardiac cells presented with 100 nM PMA increased IMP production from 4.4 +/- 0.5 (control) to 15.7 +/- 0.9 nmol/mg protein when subsequently deenergized. The effects of PMA and PE were attenuated 85 +/- 5% and 96 +/- 4%, respectively, by pretreatment of cells with 150 nM staurosporine, an inhibitor of protein kinase C. Furthermore, incubation of cardiac cells with 1 microM PMA for 24 h blunted the response to both PMA and phenylephrine 85-90%. Elevating cyclic AMP (cAMP) content to greater than 15 pmol/mg by treatment with forskolin or isoproterenol plus isobutylmethylxanthine also resulted in enhanced adenylate deaminase activity, but this stimulatory effect was not abolished by 24 h incubation with 5 microM PMA. Forskolin and PMA-induced increases in IMP production appeared to be additive. However, 0.5 microM isoproterenol inhibited the cellular response to phenylephrine by about 30% but did not affect PMA-stimulated adenylate deaminase activity. We conclude that both cAMP and protein kinase C stimulate adenylate deaminase, perhaps through selective activation of different isoforms. However, cAMP also exerts partial inhibition on alpha-adrenoreceptor-mediated increases in IMP production.     

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.     

Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination.

AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.     

Subunit structures of AMP deaminase isozymes in rat

AMP deaminases A and B have been purified to apparent homogeneity from rat muscle and liver, respectively. The molecular weights of 286,000 and 351,000 were obtained for the native muscle and liver enzymes, respectively, by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the muscle preparation exhibited a single polypeptide band with a molecular weight of 72,000; the liver preparation, a molecular weight of 85,000. The data indicate that each enzyme has a tetrameric structure.     

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 from baker's yeast. Purification and some regulatory properties.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.     

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.