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.     

The role of adenosine monophosphate nucleosidase in the regulation of adenine nucleotide levels in Azotobacter vinelandii during aerobic-anaerobic transitions

When cultures of Azotobacter vinelandii are made anaerobic the adenylate pool size remains constant or increases slightly while the adenylate energy charge decreases. Under these conditions, cell growth stops but the cells remain viable for at least 5 h with the decreased energy charge. The changes in the adenylate pool during the aerobic-anaerobic transition include: the formation of adenylates as a result of RNA degradation; the degradation of a portion of the excess AMP to form hypoxanthine by the sequential actions of AMP nucleosidase and adenine deaminase; an increase in the total adenylate pool which is stabilized at approximately 1.5 times the level in growing cells; and stabilization of the adenylate energy charge at a value near 0.3. The degradation of AMP is regulated by AMP nucleosidase, an allosteric enzyme which is activated by MgATP^2? and inhibited by P_i. The in vivo activity of AMP nucleosidase was estimated by measuring the rate of hypoxanthine formation in the culture or by measuring the activity of purified enzyme at the concentrations of AMP, ATP, and P_i found in the cells. The maximum estimated in vivo rate of AMP degradation was less than 3% of the catalytic capacity of AMP nucleosidase. Thus ample activity is present for rapid adjustments of the AMP levels in these cells. Expression of AMP nucleosidase catalytic activity is tightly controlled since high constant concentrations of intracellular AMP can be maintained for extended time periods at low adenylate energy charge values. Under these conditions controlled degradation of AMP can occur to maintain a constant AMP concentration.     

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.     

Effects of sterol manipulation on microsomal desaturase activities in Tetrahymena: with regard to thermal acclimation

The role of NH+4 ion and AMP deaminase reaction in the activation of phosphofructokinase with respect to its response to the adenylate energy charge was investigated using permeabilized yeast cells. (a) Phosphofructokinase and AMP deaminase were activated by the decrease in the adenylate energy charge. The addition of NH+4 further stimulated the phosphofructokinase activity in the presence of intracellular level of K+, and the optimal energy charge value giving the maximal response of the enzyme was shifted from 0.3 to the value above 0.5. (b) The increase in NH+4 ion produced through the activation of AMP deaminase by spermine which shows no direct action on the phosphofructokinase activity can activate phosphofructokinase with shift of the optimal energy charge value of the enzyme to 0.5 in the presence of K+, whereas the optimal energy charge value for AMP deaminase reaction was not affected by the addition of spermine. Phosphofructokinase can be activated most effectively by the physiological decrease in the energy charge under the condition of increased NH+4 in the presence of K+. The possibility that the interaction of phosphofructokinase with AMP deaminase under hypoxic condition might be a contributing factor to the Pasteur effect is discussed.     

Stabilization of the adenylate energy charge by the depletion of adenylates without glycolytic stimulation

A relationship between the AMP deaminase reaction and the recovery of the energy charge was examined using permeabilized yeast cells. Citrate inhibited the glycolytic flux and the recovery of the energy charge. The addition of spermine enhanced the recovery of the energy charge without the reversal of the inhibition of glycolysis in the presence of excess citrate. The AMP deaminase reaction can participate in the stabilization of the energy charge only by the depletion of total adenylates, and not by the glycolytic stimulation under the conditions where citrate is highly increased during aerobic growth conditions.     

AMP deaminase reaction as a control system of glycolysis in yeast. Role of ammonium ion in the interaction of phosphofructokinase and pyruvate kinase activity with the adenylate energy charge

The role of ammonium ion and AMP deaminase (EC 3.5.4.6) reaction in the activation of phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) by the decrease in the adenylate energy charge was investigated using permeabilized yeast cells. Response of AMP deaminase, phosphofructokinase, and pyruvate kinase to variation in the energy charge is typical of the ATP-regenerating enzymes: an activation with the decrease in the energy charge under the in situ conditions. The addition of polyamine activated AMP deaminase in situ, resulting in the subsequent increase in ammonium production, which can stimulate the phosphofructokinase activity with the increase in the optimal energy charge value giving maximal activity of the enzyme. The optimal energy charge value of phosphofructokinase was 0.2-0.25 in the absence of ammonium ion and was shifted to the value above 0.5 by the addition of ammonium ion, whereas Pi, an activator of the enzyme showed little effect on the increase in the optimal energy charge value. The optimal energy charge value of AMP deaminase and pyruvate kinase was not affected by the addition of their effectors. Modulation of the response to the energy charge of phosphofructokinase and pyruvate kinase was analyzed in terms of the "activation coefficient," which was defined as the ratio of the activity at the energy charge of 0.6 to that at the value of 0.9. Activation of phosphofructokinase by the physiological decrease in the energy charge (0.9 to 0.6) can be enhanced by the increase in ammonium ion specifically, although the coefficient of pyruvate kinase remained unaffected by ammonium ion. These results suggest that the AMP deaminase reaction as an ammonium-forming reaction can participate in a key role in the stimulation of phosphofructokinase or glycolytic flux in cells.     

Major determinants of purine excretion from human lymphoblasts

WI-L2 B lymphoblasts deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) excreted amounts of hypoxanthine two to three times larger than CEM T lymphoblasts deficient in HGPRT, despite similar growth rates. ATP consumption occurred at a higher rate in WI-L2 cells than in CEM cells when cultivated in a glucose-free buffer, because of higher RNA synthesis in WI-L2 cells. The introduction of actinomycin D and azaserine resulted in lower hypoxanthine excretion in WI-L2 cells than in CEM cells, not in parallel with changes of the adenylate pool size. When the energy charge was high, de novo purine synthesis was a major determinant for purine excretion. The adenylate pool ratio (AMP/ATP) change caused by the introduction of oligomycin was greater during ATP depletion and recovery in WI-L2 cells than in CEM cells. WI-L2 cells were observed to have AMP deaminase activity three to four times higher than CEM cells. The major component of AMP deaminase in these cells was liver type. The higher rate of RNA synthesis caused greater changes of (AMP/ATP) and required higher AMP deaminase activity for recovery. When the energy charge was low, AMP deaminase was a major determinant for purine excretion.     

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.     

Regulation of cytosol 5'-nucleotidase by adenylate energy charge

In the physiological range of the adenylate energy charge in liver (0.7-0.9), th rate of AMP-hydrolysis catalysed by rat liver cytosol 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) increased sharply with decreasing energy charge. In addition, a decrease in the concentration of Pi caused marked acceleration of the AMP-hydrolysing activity over the physiological range of adenylate energy charge. These responses seem to serve to protect the cells against a metabolic stress which could result from sudden utilization of ATP by removal of AMP. The AMP-hydrolysing activity of this enzyme decreased sharply as the size of the adenine nucleotide pool decreased in the physiological range. This effect may be a self-limiting response to prevent excess depletion of the pool. IMP-hydrolysing activity of this enzyme increased with increasing adenylate energy charge. But no marked response to its variation within the physiological range was observed. On the basis of the data obtained in this study, the IMP-hydrolysing activity of the cytosol 5'-nucleotidase in rat liver cells seems to be comparable to that of AMP deaminase reaction, but the AMP-hydrolysing activity was estimated to be less than 10% of AMP deaminase reaction at energy charge value of about 0.7. This strongly suggests that the AMP leads to IMP leads to inosine pathway is more significant that the AMP leads to adenosine leads to inosine pathway in rat liver.     

Effect of adenine nucleotides on the reaction catalyzed by pyruvate,orthophosphate dikinase in maize

The effect of adenine nucleotides in pyruvate, orthophosphate dikinase (EC 2.7.9.1, ATP, pyruvate, orthophosphate phosphotransferase)_was studied with the enzyme furified from maize, and with the enzyme obtained from mesophyll chloroplast extracts during assay in the direction of pyruvate conversion to phosphoenolpyruvate. (1) In studies with the purified enzyme, the relationship of initial velocity to ATP concentrations follows Michaelis-Menten kinetics, and the K_m value for ATP was 22.8 μM (± 5.1 μM, n = 5). (2) AMP was a competitive inhibitor with respect to ATP, and its K_i value was 35.8 μM (± μM, n = 4). There was no inhibition of catalysis by ADP up to a concentration of 460 μM. (3) The theoretical response of the enzyme to change in the adenylate energy charge was calculated from the kinetic constants for ATP and AMP. The experimentally obtained values were similar to the theoretical response when varying energy charge was generated by addition of appropriate amounts of ATP, ADP and AMP in assays with the purified enzyme. The response of the enzyme to energy charge at different pH values (pH 7.0, 7.5, and 8.0) was similar, although the activity of the enzyme at pH 7.0 was about 40% of that at pH 8.0. (4) When mesophyll chloroplast extracts of maize, which contain high levels of adenylate kinase, were used as the source of the enzyme and the adenylate energy charge was generated by addition of different concentrations of ATP and AMP, the influence on catalysis was similar to that with the purified enzyme. (5) The data show that the effect of varying energy chage on the activity of the dikinase is not typical of a U-type enzyme, in contrast to phosphoglycerate kinase (EC 2.7.2.3, ATP: 3-phospho-D-glycerate 1-phosphotransferase), which is more strongly regulated. (6) Evidence is presented for competition between the dikinase and phosphoglycerate kinase for ATP in mesophyll chloroplast extracts of maize. (7) When the effect of adenylate energy charge on the state of activation and the direct effect on catalysis of the dikanase are combined, the total capacity for catalysis is very dependent on the energy charge.     

The Effect of Adenine Nucleotides on Purified Phosphoenolpyruvate Carboxylase from the CAM Plant Crassula argentea

The effects of adenine nucleotides on phosphoenolypyruvate carboxylase were investigated using purified enzyme from the CAM plant, Crassula argentea. At 1 millimolar total concentration and with limiting phosphoenolpyruvate, AMP had a stimulatory effect, lowering the K_m for phosphoenolpyruvate, ADP caused less stimulation, and ATP decreased the activity by increasing the K_m for phosphoenolpyruvate. Activation by AMP was not additive to the stimulation by glucose 6-phosphate. Furthermore, AMP increased the K_a for glucose 6-phosphate. Inhibition by ATP was competitive with phosphoenolpyruvate. In support of the kinetic data, fluorescence binding studies indicated that ATP had a stronger effect than AMP on phosphoenolpyruvate binding, while AMP was more efficient in reducing glucose 6-phosphate binding. As free Mg²⁺ was held constant and saturating, these effects cannot be ascribed to Mg²⁺ chelation. Accordingly, the enzyme response to the adenylate energy charge was basically of the “R” type (involving enzymes of ATP regenerating sequences) according to D. E. Atkinson's (1968 Biochemistry 7: 4030-4034) concept of energy charge regulation. The effect of energy charge was abolished by 1 millimolar glucose 6-phosphate. Levels of glucose 6-phosphate and of other putative regulatory compounds of phosphoenolpyruvate carboxylase were determined in total leaf extracts during a day-night cycle. The level of glucose 6-phosphate rose at night and dropped sharply during the day. Such a decrease in glucose 6-phosphate concentration could permit an increased control of phosphoenolpyruvate carboxylase by energy charge during the day.     

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.     

Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation.

Adenine nucleotide breakdown to nucleosides and purine bases was measured in cultures of human lymphoblastoid cells following: 1) the inhibition of oxidative phosphorylation in the absence of glucose or 2) the addition of 2-deoxyglucose. A mutant cell line, deficient in adenosine kinase, in the presence of an adenosine deaminase inhibitor was used to measure utilization of the two pathways of AMP catabolism involving initial action of either purine 5'-nucleotidase or AMP deaminase. In such a system the appearance of adenosine induced by the oxidative phosphorylation inhibitor, rotenone, implies that approximately 70% of AMP breakdown occurs via dephosphorylation. By the same method, deamination accounts for 82% of AMP breakdown when 2-deoxyglucose is added. The occurrence of AMP dephosphorylation is not correlated with elevated concentrations of substrate or with decreased concentrations of the inhibitors of 5'-nucleotidase, ATP and ADP. Dephosphorylation occurs if, and only if, the adenylate energy charge decreases to about 0.6 in these experiments. In cultures deprived of glucose and oxygen, adenine nucleotide degradation via dephosphorylation results in recovery of normal energy charge values.     

Response of yeast pyruvate carboxylase to the adenylate energy charge and other regulatory parameters

The maximal velocity of the reaction catalyzed by partially purified pyruvate carboxylase (pyruvate + HCO₃⁻ + ATP → oxaloacetate + ADP + P_i, EC 6.4.1.1) from baker's yeast increases with an increase in the adenylate energy charge. This response is modulated by the addition of CoASAc, aspartate, or malate. Variation in energy charge does not change the apparent affinity of the enzyme for either pyruvate or HCO₃⁻, but effects on the maximal velocity may participate in regulating the rate of carboxylation of pyruvate.     

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.     

Prostaglandin E1 effects on cyclic AMP and glycogen metabolism in rat liver.

Prostaglandins E₁ or E₂ (PGE₁, PGE₂)¹ stimulated adenylate cyclase(s) from particulate fractions of whole liver homogenates 5- to 6-fold, but caused only slight (1.5- to 2-fold) stimulation of the enzyme from homogeneous hepatocytes. In contrast, glucagon stimulated enzyme from hepatocytes 12- to 15-fold and enzyme from whole liver 8- to 10-fold. Accordingly, most of the total prostaglandin-sensitive adenylate cyclase in cell suspensions was recovered in fractions containing non-parenchymal cells, and most of the total glucagon-sensitive activity was recovered with hepatocytes. PGE₁ did not change adenosine-3′,5′-monophosphate (cyclic AMP) concentrations, or alter cyclic AMP increases caused by glucagon in hepatocytes. Glucagon consistently increased hepatocyte cyclic AMP concentrations and stimulated glycogenolysis by 35 to 40%. PGE₁ did not affect basal or glucagon-stimulated glycogenolysis in the intact cells.     

Alpha 2 adrenergic amines, adenosine and prostaglandins inhibit lipolysis and cyclic AMP accumulation in hamster adipocytes in the absence of extracellular sodium

The accumulation of cyclic AMP due to adenosine deaminase plus theophylline and either isoproterenol or ACTH in the presence of adenosine deaminase plus theophylline, was inhibited by clonidine, N⁶-(phenylisopropyl)-adenosine and prostaglandin E₂. The inhibition was nearly identical in medium containing sodium ions or in medium in which sodium and its accompanying anion were substituted by an isosmotic amount of sucrose. Consistent with this, lipolysis induced by adenosine deaminase and theophylline was significantly inhibited by clonidine, N⁶-(phenylisopropyl)-adenosine and prostaglandin E₂ regardless of the presence or absence of Na⁺ in the medium. The results do not support the suggestion that extracellular Na⁺ is required for the regulation of cyclic AMP levels by hormones and neurotransmitters that inhibit adenylate cyclase.     

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.     

Comparison of AMP deaminase from skeletal muscle of acidotic and normal rats

The deamination of AMP by AMP aminohydrolase (EC 3.5.4.6) serves as the major source of ammonia production in skeletal muscle. It has been suggested that the ammonia may serve either in a buffering capacity to combat acidosis due to the accumulation of lactic acid produced during prolonged muscular activity, or as a substrate for glutamine formation which can ultimately be utilized by the kidney in adapting to metabolic acidosis. In view of this proposal, the properties of the enzyme obtained from skeletal muscle of acidotic rats have been compared with the enzyme from normal muscle. The specific activity of AMP deaminase in crude homogenates of acidotic muscle was not significantly different from normal levels. The enzyme from acidotic muscle was purified to homogeneity and was found to be identical to the enzyme obtained from normal muscle by the criteria of electrophoretic mobility, pH optimum, molecular weight, sedimentation coefficient, subunit composition, amino acid composition, monovalent cation requirement, substrate saturation, and inhibition by ATP, P_i and creatine-P. Thus, if the enzyme functions to prevent acidosis, the ability to respond to changes in the intracellular environment which accompany acidosis must be built into the structure of the enzyme normally found in skeletal muscle. Three lines of evidence strongly support this viewpoint: (a) the rate of deamination is approximately 2-fold higher at pH 6.5 than at pH 7.0, (b) the activity increases linearly with a decrease in the adenylate energy charge, and (c) within the normal physiological range of the adenylate energy charge, the enzyme is operating at only 10–20% of its maximum capacity.     

Differential response of AMP deaminase isozymes to changes in the adenylate energy charge

AMP deaminase has been prepared from white skeletal muscle fibers, red skeletal muscle fibers, cardiac muscle and liver. The isozymes from skeletal muscle, cardiac muscle and liver can be readily distinguished from one another by the shape of the adenylate energy charge response curve. However, the enzyme prepared from different skeletal muscles which consists of predominately red or white fibers are indistinguishable from one another by this criterion.