Changes in AMP deaminase activities in the hearts of diabetic rats

AMP deaminase from normal and diabetic rat hearts was separated on cellulose phosphate and quantitated by HPLC. From soluble fractions three different AMP deaminase activities, according to KCl elution from cellulose phosphate and percent of total activity were: 170 mM (85%), 250 mM (8%) and 330 mM (7%) KCl. The AMP deaminase activity which eluted with 170 mM KCl was resolved to two distinct peaks by HPLC anionic exchange. After 4 weeks of diabetes the heart enzyme profile change to: 170 mM (10%), 250 mM (75%) and 330 mM (15%). Once purified the four activities were kinetically distinct: 170 mM KCl cytosolic, AMP Km = 1.78, stimulated by ATP, GTP, NADP and strongly inhibited by NAD; 170 mM KCl mitochondria AMP Km = 17.9, stimulated by ATP, ADP; 250 mM KCl isozyme, AMP Km = 0.66, stimulated by ADP; and 330 mM KCl isozyme, AMP Km = 0.97, inhibited by ATP, NAD(P).     

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.     

AMP deaminases of rat small intestine

Phosphocellulose column chromatography revealed the existence of two forms of AMP deaminase both in whole tissue and in the intestinal epithelium. AMP deaminase I, which eluted from the column as a first activity peak, exhibited hyperbolic, nonregulatory kinetics. The substrate half-saturation constants were determined to be 0.3 and 0.7 mM at pH 6.5 and 7.2, respectively, and did not change in the presence of ATP, GTP and Pi. AMP deaminase II, which eluted from the column as a second activity peak, was strongly activated by ATP and inhibited by GTP and Pi. The S0.5 constants were 3.5 and 7.1 at pH 6.5 and 7.2, respectively. At pH 7.2 ATP (1 mM) S0.5 decreased to 2.5 mM and caused the sigmoidicity to shift to hyperbolic. The ATP half-activation constant was increased 9-fold in the presence of GTP and was not affected by Pi. Mg2+ significantly altered the effects exerted by nucleotides. The S0.5 value was lowered 10-fold in the presence of MgATP and 5-fold in the presence of MgATP, MgGTP and Pi. When MgATP was present, AMP deaminase II from rat small intestine was less susceptible to inhibition by GTP and Pi. A comparison of the kinetic properties of the enzyme, in particular the greater than 100% increase in Vmax observed in the presence of MgCl2 at low (1 mM) substrate concentration, indicates that MgATP is the true physiological activator. GuoPP[NH]P at low concentrations, in contrast to GTP, did not affect the enzyme and even activated it at concentrations above 0.2 mM. We postulate that AMP deaminase II may have a function similar to that of the rat liver enzyme. The significance of the existence of an additional, non-regulatory form of AMP deaminase in rat small intestine is discussed.     

GTP,as well as ATP,can act as a substrate for the intrinsic protein kinase activity of synaptic plasma membranes

Summary GTP as well as ATP can act as phosphate donor for the intrinsic protein kinase activity of synaptic plasma membranes. There are many similarities between the activities observed with ATP or GTP. Both need a divalent cation, Mg²⁺ being preferred, both are slightly inhibited by Na⁺, and more strongly by K⁺, both are inhibited by theophylline and adenosine. The K_m for GTP (0.13 mM) is similar to that ATP (0.12 mM). There are, however, some differences in properties. When GTP instead of ATP is the phosphate donor the pH optimum is 6.5 instead of 7.4. In addition NH ₄ ⁺ inhibits the transfer of phosphate from GTP but not from ATP. More importantly, cyclic AMP only stimulates the transfer of phosphate from ATP not from GTP. SDS gel electrophoresis reveals that similar membrane proteins are phosphorylated by GTP and ATP in the presence or absence of cyclic AMP. This suggests that there may be two different types of protein kinase in the synaptic plasma membrane which act on similar membrane proteins. One is stimulated by cyclic AMP and is specific to ATP while the other is unaffected by cyclic nucleotides and can use either ATP or GTP as phosphate donor.Deceased     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

Effects of pyrophosphate, triphosphate, and potassium chloride on adenylate deaminase from rat muscle

Inorganic pyrophosphate and triphosphate inhibit adenylate deaminase from rat skeletal muscle with K1 values of 10 and 1.5 microM, respectively, in the presence of 150 mM KCl at pH 7. They act by reducing the apparent affinity of the enzyme for AMP, with relatively small effects on Vmax. The inhibitions are diminished by H+, the KI values increasing two- to threefold in going from pH 7.0 to 6.2, and are relieved by ADP. These properties are similar to the inhibitions produced by GTP and ATP, indicating that pyrophosphate and triphosphate act like analogues of the nucleoside triphosphates. Neither of these inhibitors shows relief of inhibition at high concentrations as do ATP and GTP. These results suggest that nucleotides interact with the inhibitor site of the enzyme primarily through their phosphate moieties and with the activator site primarily through their nucleoside moieties. As the concentration of KCl is increased from 25 to 300 mM, the apparent affinities of the enzyme for ATP, GTP, orthophosphate, pyrophosphate, and triphosphate are decreased 8-100-fold. The cooperativity of the inhibitions is increased with the Hill coefficient rising from 1.0 to 1.3-1.8, and the maximum inhibition approaches 100%. Maximum activation by ADP is reduced from 1800% at 25 mM KCl to 80% at 200 mM KCl. Experiments with (CH3)4NCl indicate that activation of the enzyme by KCl involves both specific K+ effects and ionic strength effects.     

Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots

A membrane-bound, monovalent cation-stimulated ATPase from Zea mays roots has been purified to a single band on sodium dodecyl sulfate gel electrophoresis. Microsomal preparations with K⁺ -stimulated ATPase activity were extracted with 1 m NaClO₄, and the solubilized enzyme was purified by chromatography on columns of n-hexyl-Sepharose, DEAE-cellulose, and Sephadex G-100 Superfine. A 500-fold purification over the activity present in the microsomes was obtained. The K⁺ -stimulated activity shows positive cooperativity with increasing KCl concentrations. The purified enzyme shows K⁺ -stimulated activity with ATP, GTP, UTP, CTP, ADP, α + β-glycerophosphate, p-nitrophenyl phosphate, and pyrophosphate as substrates. Under most conditions ATP is the best substrate. Although dicyclohexyl carbodiimide and Ca²⁺ inhibit and alkylguanidines stimulate the K⁺ -ATPase while bound to microsomes, they have no effect on the purified enzyme.     

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.     

Isolation and regulation of piglet cardiac AMP deaminase

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a K_m of 2 mM for 5-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the K_m of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD⁺, whereas, the K_m was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in V_max with no change in K_m. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.     

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.     

Etude de quelques propriétés de la phosphofructokinase érythrocytaire de l'homme normal

Human erythrocyte phosphofructokinase was purified 150 fold by DEAE cellulose adsorption and ammonium sulfate precipitation.At pH 7,5 the enzyme exhibits allosteric kinetics with respect to ATP, fructose 6 phosphate, and Mg²⁺.ATP at high concentration acted as an inhibitor and ADP, 5′AMP, 3′,5′, AMP, acted as activators. Both effectors seemed to decrease the homotropic interactions beetween the fructose 6 phosphate molecules.The activators increased the affinity of phosphofructokinase for the substrate (F6P), the inhibitor decreased it.These ligands had no effect on the maximum velocity of the reaction except in the case of ADP.Interactions between the substrates and the effector ligands on the enzyme were considered in terms of the Monod - Changeux - Wyman model for allosteric proteins.With GTP and ITP, no inhibition was observed. At saturing concentration of GTP, ATP still inhibited phosphofructokinase.Both 3′5′ AMP and fructose 6 phosphate increased the concentration of ATP required to produce an inhibition of 50 %.Citrate, like ATP, inhibited phosphofructokinase by binding most likely at the same allosteric site. Erythrocyte phosphofructokinase is inhibited by 2–3 DPG.The study of the relation log V max = f (pH) suggested, that the active center contains at least one imidazole and one sulfhydryl group.     

Purification and partial characterization of an AMP deaminase from the marine invertebrate Palaemon serratus

• 1.1. AMP deaminase from Palaemon serratus tail muscle was partially purified by chromatography on cellulose phosphate.
• 2.2. Muscle homogenates expressed very low enzyme activities and the presence of ATP was necessary to detect AMP deaminase. The specific activity and substrate affinity of the purified enzyme were also very low.
• 3.3. The purified prawn muscle AMP deaminase was contaminated by contractile proteins, one of the major contaminants being actin.
• 4.4. The enzyme displayed a very high affinity for actomyosin which was only partially abolished by pyrophosphate.

     

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.     

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.     

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+.     

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.     

Human erythrocyte 5'-AMP aminohydrolase. Purification and characterization

Human erythrocyte 5'-AMP aminohydrolase has been obtained using phosphocellulose chromatography and affinity chromatography on a GTP-agarose column to yield a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 285,000, and is comprised of four subunits. Since limited quantities of the homogeneous enzyme were available, the kinetic properties of a nonhomogeneous preparation purified about 20,000-fold over the red blood cell lysate by phosphocellulose chromatography were examined. Like the muscle enzyme, it exhibits a sigmoid AMP saturation curve in the absence of activating monovalent cations; a hyperbolic saturation curve is observed in the presence of 0.15 M KCl. Activation by monovalent cations and ATP, and inhibition by Pi, PPi, GDP, GTP, and 2,3-diphosphoglyceric acid were studied in more detail.     

Purification and properties of a glycoprotein adenosine 5′-monophosphatase from the plasma membrane fraction of Arachis hypogaea cotyledons

An adenosine 5′-monophosphatase (AMPase) has been purified from the plasma membrane fraction of germinating cotyledons of peanut (Arachis hypogaea L.) by selective solubilization of the membrane-bound enzyme with 0.5% n-octyl β-glucoside at a protein-to-detergent ratio of 2:3 in the presence of Mg²⁺ and EDTA, followed by ion exchange chromatography on DEAE-cellulose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it showed a single protein band with a molecular weight of 55 000. The enzyme is a glycoprotein with 42.7% carbohydrate content. It had a broad pH optimum of 5.0–6.0. The K_m and V_max values were 1.08·10⁻³ M and 8.5 μmol/min per mg protein, respectively, with 5′-AMP as substrate. The enzyme is specific for 5′-AMP. Other nucleotides (GMP, UMP, CMP, ADP, GDP, ATP, GTP and UTP) as well as phosphorylated sugars were not hydrolyzed. p-Nitrophenyl phosphate was hydrolyzed at a relatively much lower rate (15%) and the substrate affinity (1/Km was only one-tenth that of AMP. The purified enzyme is competitively inhibited by ADP (K_i = 2.4 mM) and is also inhibited by NaF in a non-competitive manner with a K_i value of 35 mM. Divalent cations, Ca²⁺, Mg²⁺, Hg²⁺, Zn²⁺, Mn²⁺, Ni²⁺ and monovalent cations, K⁺, Li⁺ and Na⁺ had no effect on the enzyme activity. The purified enzyme was highly unstable, losing its total activity within 24 h at −20°C, or 0°C, while under these conditions the unpurified solubilized enzyme (octyl glucoside extract) was stable for several days, indicating that some stabilizing factors, most likely phospholipids, were lost during the enzyme purification.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

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.