S37
The Purine Nucleotide Cycle Revised I M Lowenstein Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254, USA
Summary
NH3GDP
+ pi Aspartate + GTP
Key words
adenosine adenylate inonophosphate, deaminase, adenylosuccinase, adenyloscuccinate, adenyloscuccinate lyase, adenylosuccinate synthetase, deamination, exercise, fluoroanalogs, inosine monophosphate, isozymes, phosphofructokinase, phosphorylase, purine nucleotide cycle, reamination
Fig. 1 The purine nucleotide cycle. The reactions shown are catalyzed by a, adenylate deaminase; b, adenylosuccinate synthetase; and c, adenylosuccinate yase (adenylosuccinase).
tate to ammonia and fumarate occurred by a cyclical process
which was termed the purine nucleotide cycle (Fig. 1) (36).
Introduction The sum of reactions 1,2, and 3 yields:
The relationship between muscular work and the production of ammonia was studied in the laboratories of Parnas and Embden in the late 1920s (21—23, 46—48). Both groups recognized that the adenylate deaminase reaction (68) is the major source of ammonia in muscle:
AMP +1120 — IMP + NH3
aspartate + GTP —+ fumarate + NH3 + GDP + P1
(4)
One turn of the cycle results in the deamination of one molecule of aspartate at the expense of the cleavage of one molecule of GTP.
(1)
Reaction I is not reversible under physiological conditions. It was concluded that other reactions must exist for converting IMP back to AMP. These reactions were not identified until 1955 (36):
IMP + asparate + GTP —+ adenylosuccinate + GDP + P(2) (3) adenylosuccinate AMP + fumarate Reactions 2 and 3 are catalyzed by adenylosuccinate synthetase and adenylosuccinate lyase (adenylosuccinase), respectively. In 1971 it was shown that extracts of rat skeletal
muscle can produce ammonia from aspartate under condi-
Subsequently it was shown that the purine nucleotide cycle operates in hind limb of rat during strenuous work. The hind limb was investigated both in vivo and with perfused preparations. Exercise was induced by electrical stimulation. Adenylosuccinate was not present in detectable amounts in resting muscle, but it appeared and rose sharply during exercise to reach a maximum of 21 nmol/g dry weight of muscle. It declined again during recovery from exercise. IMP increased from resting values of about 0.1 .tmol/g dry weight to about 9 mol/g dry weight during strenuous exercise (6 square-wave electrical pulses of 10 ms duration/s) and then declined during recovery. Smaller increases in IMP were observed when the stimulation was decreased to I pulse of 10 ms duration/s, or when resting muscle was perfused with epinephrine, or was made hypoxic. Ammonia increased stoi-
tions that cause the consumption of ATP, i. e., conditions that mimic muscle doing work (38, 80). The conversion of aspar-
chiometrically with IMP (30, 37). These papers also presented measurements of various other metabolites, including certain amino acids, during rest and exercise, as well as calculations of
Int.J. Sports Med. 11(1990) S37—S46
the intracellular concentrations of free ADP and AMP. A
Georgmieme Verlag StuttgartNewYork
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This review is restricted to the operation of the purine cycle in mammalian muscle. A previous review provided a summary of early evidence for the operation of the cycle and of various functions proposed for the cycle. It also provided a brief history of work on ammonia production by muscle and other tissues and of the discovery of the enzymes of the purine nucleotide cycle (36). Recent reviews on the purine nucleotide cycle include those of Terjung et al. (78) and van Waarde (86).
S38 mt. .J. Sports Med. 11(1990)
.1. M Lowenstein
monia as a function of time.
The results obtained with the exercising rat hind limb were interpreted as evidence for the occurrence of the purine nucleotide cycle in skeletal muscle. The following scenario accounts for the experimental observations. During intense exercise, the rate of ATP utilization exceeds the capacity of oxidative phosphorylation and glycolysis to regenerate ATP. As is discussed below, the tendency for this to occur is much greater in muscles with a low than with a high aerobic capacity. The creatine phosphate reservoir acts as a buffer which maintains a high ATP concentration via the creatine kinase reaction: creatine
phosphate + ADP creatine + ATP
(5)
Reaction 5 is assumed to be in equilibrium at rest and during exercise because of the high activity of creatine
kinase found in skeletal muscle. However, NMR measurements with frog gastrocnemius indicate that some deviation from equilibrium may occur during exercise (12). If the capac-
ity of the creatine phosphate/creatine buffer is exceeded, in other words, if most of the creatinephosphate is used up, ADP formed during muscle contraction rises. This leads to an increased formation of AMP via the myokinase reaction:
2 ADP+AMP+ATP
(6)
Reaction 6 is also assumed to be in equilibrium
under all physiological conditions. This assumption is justified by the high activity of myokinase in skeletal muscle, but has not been tested experimentally. Thus, ADP and AMP rise in concert, with AMP rising as the square of the ADP concentration. ADP is the most effective activator of adenylate deaminase prepared from hind limb of rabbit. Moreover, lactic acid accumulates during intense muscular activity. The resulting acidification also serves to activate adenylate deaminase because the enzyme has pH optimum of about 6.2 (87).
Activation of adenylate deaminase leads to the removal of AMP, with the result that reaction 6 is pulled to the right until the ATP/ADP ratio is high:
The functions proposed for the purine nucleotide cycle (36) are here presented in slightly recast form: 1. It serves to regulate the relative concentrations of AMP, ADP, and ATP. In particular it serves to maintain a high ATP/ADP ratio. Insofar as AMP, ADP, and ATP act as substrates, activators, and inhibitors of many reactions, it acts as a general, high level metabolic control mechanism. 2. It is a pathway for the replenishment of citric acid cycle intermediates by generating fumarate from aspartate. 3. It is a pathway for the production of ammonia from amino acids; put differently, it is a pathway
that makes possible the deamination of various amino acids for energy production. Ammonia serves in a small way to diminish the drop in pH caused by lactic acid production. 4. It assists in the regulation of phosphofructokinase activity and hence of glycolysis by regulating the AMP concentration; AMP being an activator of phosphofructokinase. (This is a specific example of the general control mechanism mentioned in 1.) 5. It may serve in the regulation of phosphorylase b by generating IMP during severe exercise while keeping AMP low. Evidence for these proposed functions will now be examined briefly. 1. The maintenance of a high ATP/ADP ratio by the combined action of myokinase and adenylate deaminase cannot be demonstrated by direct metabolite measurements. The ADP and AMP contents of rat hind limb show little or no change between rest, exercise, and recovery from exer-
cise, being 0.60 and 0.11 tmol/g wet weight for ADP and AMP, respectively. Skeletal muscle actin contains one tightly bound molecule of ADP per actin monomer. The binding is so tight that the bound ADP is not available to the pool of free ADP. The content of actin monomer is about 0.6 mol/g wet weight of muscle (41), which is about the same as the total ADP
content. It follows that virtually all of the ADP is bound and unavailable. The standard error of these measurements is too great to use total and bound ADP to calculate free ADP. For this reason, free ADP and AMP were calculated using the assumption that the creatinekinase and myokinase reactions are in equilibrium. The calculated contents of free ADP and AMP in resting hind limb of rat were 19 and 0.12 nmol/g wet weight, respectively, at pH 7.0. After 15 mm of exercise, these rose to 36 and 0.56 nmol/g wet weight, respectively, at pH 6.6. According to these calculations, free ADP accounts for only 3.2%
and 5.4% of the total ADP in muscle at rest and during
(6) (1)
vigorous exercise, respectively. Similarly, free AMP accounts for only 0.11% and 0.50% of the total AMP, respectively. Unlike ADP, there seems to be no protein to which most of the AMP binds tightly. Possibly the AMP is stored in a compart-
(7)
ment where it is not available to myokinase or adenylate deaminase (30). More detailed calculations of adenine nu-
Adenylate deaminase and myokinase are
cleotide contents during rest and exercise were presented by Lowenstein and Goodman (37). The calculated contents of free AMP of 0.12 and 0.56 .tmol/g wet weight correspond to about 0.24 and 1.1 p.M AMP. These concentrations are far below optimum for adenylate deaminase. At 0.1 and 1.0 p.M AMP, adenylate deaminase in rat hind limb can deaminate
AMP +
AlP
AMP + H20 — IMP +
NH3
2ADP+ H20 —+ ATP +
IMP +
2ADP Sum:
Functions of the Purine Nucleotide Cycle
NH3
among the most active enzymes of skeletal muscle and their combined action explains the formation of IMP during severe exercise. IMP accumulates because adenylate deaminase occurs in far higher activity than adenylosuccinate synthetase and adenylosuccinase; in other words, the reactions that convert IMP back to AMP cannot keep up with the rate of IMP formation. The question of whether or not the reactions of the purine nucleotide cycle operate concurrently is dealt with below.
AMP at maximum rates of 0.02 and 0.2 p.mol/g wet weight/mm, respectively. These rates are substantially greater than the observed rates of IMP formation. This implies that adenylate deaminase is not fully activated under these conditions (30).
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detailed discussion of these data is outside the scope of this review. The stoichiometric increase in ammonia and IMP indicates that ammonia diffuses only slowly from muscle. A similar conclusion was reached by Rosado et al. (58), who injected rats with NH4C1 and studied the resulting distribution of am-
The proposed function of maintaining a high ATP/ADP ratio is therefore supported by calculations of metabolite contents. Verification independent of such calculations is highly desirable. This would probably require determination of adenine nucleotide and other metabolite contents in different compartments of the muscle cell; techniques for carlying out such measurements are becoming available. 2. The contents of citric acid cycle intermediates were measured in rat hind limb during rest, exercise, and recovery from exercise. The contents of fumarate and malate increased four-fold and the sum of the contents of all citric acid cycle intermediates doubled after 10 mm of exercise. All metabolites returned to resting values during recovery from exercise. These observations are consistent with the proposal that the purine nucleotide cycle serves to rep1enish citric acid cycle intermediates. Hadacidin (N-formylhydroxyaminoacetate) is a competitive inhibitor with respect to aspartate in the adenylosuccinate synthetase reaction. Accumulation of IMP in rat
hind limb during 10 mm of exercise was 1.5 and 2.5 mol/g dry weight in the absence and presence of hadacidin, respectively. The difference of 1.0 imol represents the partial inhibi-
tion of adenylosuccinate synthetase by hadacidin. The increase in fumarate plus malate over the same interval was 1.14 and 0.54 .tmo1/g dry weight, respectively. Thus, hadacidin inhibited fumarate plus malate production by 53%. Hadacidin has no effect on the levels of IMP, fumarate, and malate in unexercised controls. These experiments constitute strong evidence that part, if not all, of the increase in citric acid cycle intermediates observed during exercise is due to the operation of
the purine nucleotide cycle. Other pathways for increasing citric acid cycle intermediates do not seem to operate in skeletal muscle (4, 5). Terjung et al. (78) believe that these data cannot be interpreted to show that operation of the purine nucleotide cycle serves to increase citric acid cycle intermediates in contracting muscle because the hind limbs used by us were composed of FG and FOG fibers. They fail to provide an alternative explanation for the higher accumulation of IMP and for
the inhibition of fumarate plus malate production in the presence of hadacidin which we observed during the operation of the cycle, regardless of whether the fibers were mixed or not. They also fail to provide alternative interpretations for the accumulation of malate and fumarate during the operation of the cycle in the absence of hadacidin. In mixed muscle, for a given total activity, if one fiber type shows low activity of the purine nucleotide cycle, then the other fiber type must show a correspondingly higher activity. Oddly, in their own paper they conclude that increased IMP accumulation in the presence of hadacidin in mixed muscle is strong evidence that turnover of the purine nucleotide cycle occurred (44). These authors showed that FG fibers of plantaris (50 %—55 % FG, 40%—45 % FOG) were the major site of purine nucleotide cycle activity.
AlCAriboside has been used to partially disrupt the purine nucleotide cycle at the level of adenylosucci-
nate synthetase and adenylosuccinase, while leaving the adenylate deaminase reaction to function (27). Treatment with AlCAriboside results in a pronounced decrease in tension both with single, non-tetanizing pulses and with tetanic stimulation (a detailed explanation of the mode of action of this compound is given under Adenylosuccinas). During both aerobic and anaerobic stimulation of gastrocnemius, adeny-
mt. J. Sports Med 11(1990) S39 late deaminase in AlCAriboside-treated animals was more active and adenylosuccinate synthetase and adenylosuccinase were less active than in controls. Total energy production was impaired in the test group, but lactate production was similar
in test and control groups. The authors concluded that the purine nucleotide cycle plays an anaplerotic role in providing citric acid cycle intermediates that enhance energy production in contracting skeletal muscle. Total citric acid cycle intermediates were not determined in this study, but changes in malate
and citrate in response to exercise were similar in salinetreated and AlCAriboside-treated animals. The decrease in tension may therefore be due to other effects of AlCAriboside or its metabolic products (see Adenylosuccinase).
The original demonstration of the operation of the purine nucleotide cycle was performed with a soluble fraction obtained from skeletal muscle (cytosol) (38, 80). Skeletal
muscle cytosol and mitochondria were prepared separately and combined to demonstrate that the generation of fumarate by the purine nucleotide cycle in the cytosol can serve to accelerate the oxidation of pyruvate via the citric acid cycle in the mitochondria. In this system, the acceleration of pyruvate oxidation depended on the addition of GTP, aspartate, and AMP or IMP (69).
The functioning of the purine nucleotide cycle was examined further in particle-free extracts of gastrocnemius and soleus. The activities of the enzymes of the cycle increased in parallel as the ATP/ADP ratio was lowered from 50 to I, i. e., a low energy state leads to an activation of the operation of the cycle. Interestingly, the inhibition of adenylosuccinate synthetase by high concentrations of IMP (see below) was
not observed by these workers. Adenylosuccinate (0.6 mM) activated adenylate deaminase two-fold (39). The adenylosuccinate concentration of 0.6 mM is much higher than the maximum reported in skeletal muscle. It is therefore unlikely that adenylosuccinate is a physiological activator of adenylate deaminase.
3. Ammonia production during muscle contraction has been demonstrated repeatedly (7, 34, 90, to mention only a few recent papers on this topic involving human subjects). The best evidence that the ammonia arises via the deamination of AMP is the observed stoichiometry between IMP and ammonia production. Evidence that glutamate dehydrogenase is not involved in ammonia production by exercising muscle rests on a comparison of its activity in the direction of deamination with the observed rates of ammonia production (30, 37). The best evidence of the involvement of the purine nucleotide cycle in the deamination of amino acids in skeletal muscle was obtained with 15N-labelled leucine. Depending on when it was provided to the exercised, perfused hind limb, between 14% and 24% of the amino nitrogen used in the reamination of IMP originated from the 15N-labelled amino acid (31). 4. The regulatory properties of phosphofructokinase are complex. The enzyme is inhibited by physiological concentrations of ATP and by citrate when ATP is in the inhibitory range. The ATP inhibition is relieved by its degradative products, namely orthophosphate, ADP, and AMP. Other
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The Purine Nucleotide Cycle Revised
J. M Lowenstein
S40 mt. I Sports Med. 11(1990)
bition, are fructose-6-phosphate (F6P), fructose-i ,6-diphosphate (Fl ,6diP), glucose-i ,6-diphosphate (G 1 ,6diP), cAMP (83), and fructose-2,6-diphosphate (F2,6diP). The activation by Fl,6diP is enhanced synergistically by AMP (81). F2,6diP was not available in 1976; it was shown subsequently that synergism also occurs between F2,6diP and AMP (84, 85). ADP is a weak activator, but its effect is increased greatly by
orthophosphate (49). The AlP inhibition increases with decreasing pH. For example, with 5 mM ATP, the Kapp for F6P is increased by a factor of 50 as the pH is lowered from 7.7
to 6.8. In other words, lowering the pH diminishes the apparent affinity of the enzyme for F6P. This effect is abolished
by 10 .tM F2,6diP. Other effectors are not as powerful as F2,6diP, but combinations of AMP and Gl,6diP are as effective as F2,6diP (18). Most studies of the regulatory behaviour of phosphofructokinase were conducted at enzyme concentrations well below those which occur in the muscle cell. When the enzyme is used at physiological concentrations, or in the presence of ethylene glycol, which favors aggregation of the dilute enzyme, the ATP inhibition is greatly diminished. However, activating effectors, such as AMP and orthophosphate, still work by increasing the apparent affinity of the enzyme for F6P (10). F2,6diP is the most powerful activator of phosphofructokinase. Its concentration in resting skeletal muscle is very low and actually decreases during exercise. It is therefore difficult to assign a regulatory role for F2,6diP in glycolysis during muscle contraction, although it may have such a role in resting muscle in response to epinephrine (33). Studies of the oscillatory behaviour of glycolysis in cell-free extracts of rat skeletal muscle show that F2,6diP is not involved in the control of the oscillations. If 0.5 .tM F2,6diP is added to the extracts,
the oscillations are abolished, but 0.1 .LM F2,6diP does not prevent the oscillations. In the presence of physiological concentrations of ATP and citrate, physiological concentrations of F2,6diP do not block the oscillations, but increase their
frequency. The physiological role of F2,6diP in skeletal muscle remains uncertain. Its concentration in vivo is only about 8% of that of phosphofructokinase subunits. Moreover, appropriate changes in F2,6diP have not been observed upon initiation of muscular exercise (79).
5. Activation of phosphorylase b by IMP and AMP was first reported by Con et al. (17), but a physiological role for IMP was ruled out because IMP is a much weaker activator than AMP. Activation of the enzyme by IMP was suggested as a mechanism that could account for the high rates of glycolysis observed in mice deficient in phosphorylase kinase (ICR/IAn mice). AMP changes little during exercise in such mice, but IMP rises more rapidly and to higher levels than in normal mice. These observations were interpreted as showing that IMP can act as an activator of phosphorylase b not only in muscle of ICR/IAn mice, but also in muscle of normal mice
was below control levels (6). IMP accumulated rapidly in exercising muscle, reaching 1,200 .tM after 10 s, while the (calcu-
lated) AMP concentration was around i tM. At this point, maximum conversion to the a form of phosphorylase had occurred. The accumulated IMP was sufficient to activate the a form substantially. After 20 s of exercise, IMP had reached 1560 .LM and AMP around 4 .tM. This IMP concentration was sufficient to activate phosphorylase b about six-fold over the basal activity, whereas the AMP concentration was insufficient to activate the enzyme. A six-fold activation of the b form was not sufficient to account for the observed rates of glycolysis; it was speculated that other activators may occur in muscle which may act in concert with IMP (6). Rahim et al. (56) observed a 40% decline in phosphorylase a in mouse hind limb between 5 and 60 s of exercise. Reversal of the phosphorylase b
to a conversion, despite continued contractile activity, was also studied by Conlee et al. (16). These workers found that the
conversion was transient in both plantaris (41 % FG, 53% FOG, and 6% SO) and soleus (15% FOG and 85% SO). In summary, kinetic studies with muscle extracts and the purified enzyme, as well as measurements of changes in IMP contents
during exercise, show that IMP can act as an activator of phosphorylase b. Under these conditions the AMP concentration is too low to be effective as an activator. Activation of the b
form by IMP does not appear to be sufficient to account completely for the observed rates of glycogenolysis, but IMP may act in concert with other, unidentified activators. Concurrent Versus Segmented Operation of the Cycle
As has already been mentioned, IMP accumulates during vigorous exercise because adenylate deaminase (reaction i) operates at higher rates than adenylosuccinate synthetase (reaction 2) and adenylosuccinase (reaction 3); in other words, the reactions that convert IMP back to AMP cannot keep up with the rate of IMP formation. The question has been raised whether reactions 2 and 3 operate at all during the phase when IMP is being formed by reaction 1 (43, 44, 78). Hadacidin inhibits adenylosuccinate synthetase (see above). In the exercising hind limb, a greater accumulation of IMP occurred in the presence of hadacidin than in its absence within 1 mm after the onset of exercise and thereafter. Maximum accumulation on IMP occurred 10 mm after starting the exercise. It follows that reaction 2 must have been functioning long before maximum accumulation of IMP had occurred (5). Other evidence showed that adenylosuccinate rises and reaches near maximum levels during exercise (30, 37). It was concluded that the cycle operates even while AMP is being deaminated. It is not known at which point during or after exercise reactions 2 and 3 reach their maximum rates. It seems likely that this will depend on the type of muscle being studied. Red Versus White Muscle
(55, 56).
Comparison of the activation of phosphorylase b by AMP and IMP in vitro showed that it was necessary to use 50 times as much IMP in order to obtain the same degree of activation as is obtained with AMP (6). Assays of the activities
of phosphorylase a and b during exercise in hind limb showed that the conversion of the b to the a form reached a maximum
within 10 s after the start of exercise. However, after 20 s phosphorylase a had declined considerably, and by 1 mm it
Skeletal muscle fibers have been classified into three types on the basis of their biochemical and physiological properties, namely fast-twitch-glycolytic (FG), fast-twitch-oxidative-glycolytic (FOG), and slow-twitch-oxidative (SO) (8, 50). Earlier terminologies for different fiber types were compared by Peter et al. (50). For a detailed discussion of skeletal muscle fiber types, their function, and their enzyme profiles,
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activators, all of which appear to act by relieving the ATP inhi-
mt. J. Sports Med. 11 (1990) S41
The Purine Nucleotide Cycle Revised
Different skeletal muscle fiber types contain different activities of adenylate deaminase. Raggi et al. (53) showed that white muscles of rabbit had much higher adenylate deaminase, myokinase, and creatine kinase activities than red muscles. These authors also examined red and white muscles of rat and pigeon, with similar results, Gerez and Kir-
even though more intense stimulation was employed. Dudley and Terjung (19) developed these findings further. They re-
ported that upon moderately intense electrical stimulation, FG muscle fibers ("white" fibers) showed a decrease in total adenine nucleotides by as much as 50%, depletion of their creatine phosphate, and severe acidosis. Under the same conditions, the FOG fibers of the same muscle ("red" fibers) showed little depletion of adenine nucleotides, only a partial decrease in creatine phosphate, and much less acidosis. Elimi-
sten (29) had shown previously that muscles poor in mitochondna, such as leg muscle of rat, produce more ammonia during exercise than do muscles rich in mitochondria, such as breast
nation of the blood flow before initiating the contractions abolished the distinction between the FG and FOG fibers.
muscle of pigeon. Differences in adenylate deaminase and
chiometric loss of ATP. Dudley and Terjung (20) concluded that cellular acidosis is a major factor in causing activation of adenylate deaminase, especially in FG fibers. Whitlock and
adenylosuccinate lyase activities of different fiber types of rat
skeletal muscles were documented by Winder et al. (91). Adenylate deaminase activity was highest in white quadriceps
(mainly FG), next highest in red quadriceps (mainly FOG), and lowest in soleus (SO). The activities were in the ratio 100:84:40, respectively. It should be pointed out that in each muscle adenylate deaminase activity measured under conditions of Vmax is present in great excess over the deaminase ac-
tivity actually expressed in intact muscle. In other words, adenylate deaminase in vivo operates at only a small fraction of its Vmax. A significant correlation was observed between
adenylate deaminase and phosphofructokinase activities (r=0.97).
Both now produced a high amount of IMP and a stoi-
Terjung (89) showed subsequently that during prolonged ischemic stimulation even soleus (SO) undergoes a large depletion in adenine nucleotides, which can be accounted for
by the accumulation of IMP. The authors believe that the limited depletion of the adenine nucleotide pool that is observed during exercise in muscles such as soleus is due in part to the limited accumulation of lactic acid, because when conditions are used which do lead to a large accumulation of lactic acid, such as prolonged stimulation during ischemia, a large decrease in ATP and a stoichiometric increase in IMP does occur.
The abundance of adenylate deaminase
Adenylate Deaminase Structure and Expression
mRNA in different skeletal muscle fiber types of rat was assessed by Sabina et al. (61). The mRNA was most abundant in outer gastrocnemius (FG), less abundant in inner gastrocne-
The complementary DNA (cDNA) of adenylate deaminase messenger RNA (mRNA) has been cloned,
mius (FOG), and least abundant in soleus (SO), and paral-
and the amino acid sequence of the enzyme deduced (61). The
lelled the differences in adenylate deaminase activity found by Raggi et al. (53) and Winder et al. (91).
enzyme has a molecular weight of about 80,000. Three
Treadmill training for 12 weeks resulted in a 29% decline in adenylate deaminase activity in red quadriceps, but resulted in no change in the other types of muscle. Adenylosuccinase activity was highest in red quadriceps and approximately equal in white quadriceps and soleus. Its activity remained unchanged during the exercise regimen (91).
Cat biceps brachii (>75% FG) and soleus
(>92% SO) were analyzed by NMR. The creatine phosphate/creatine ratios calculated from the data so ob-
isoforms of adenylate deaminase have been identified during skeletal muscle development. An embryonic isoform appears in the hind limb of rat between 7 and 14 days of gestation; this form is also expressed in non-muscle tissues in the perinatal and adult rat. A second form appears 4—6 days before birth and persists for 2—3 weeks after birth; this form is expressed only in
skeletal muscle. The adult isoform appears after birth, and reaches maximum levels after 3 weeks; this form, too, is expressed only in skeletal muscle (40). The expression of these three, stage-specific transcripts during development has been studied further in myocytes in culture and in hind limb of rat (62).
tained were 175 in the FG and 2.1 in the SO muscles. The calcu-
lated ADP concentration in FG was 0.3 tM and in SO was 14 1XM. Intracellular orthophosphate was 3 mM in FG and 10
mM in SO. The intracellular pH was 7.0 in both types of muscles (42). Similar measurements were presented for rat hind limb, except that in this study the limb was subjected to exercise (35). IMP formation could not be quantitated in this
Intracellular Localization of Adenylate Deaminase
Adenylate deaminase has been shown to be at-
tached to the A band of the sarcomere (3). In solution, the
Meyer and Terjung (43) showed that during
muscle enzyme forms a tight complex with heavy meromyosin and with subfragment-2 of heavy meromyosin. Subfragment-2 is believed to be involved in the interaction of myosin and actin (1). The complex formed between pure HMM and adenylate
tetanic stimulation of rat muscles in situ, the total adenine nucleotide content of gastrocnemius (FG and FOG) fell by as much as 50%, whereas that of soleus (SO) fell by only 10%. In gastrocnemius the fall in adenine nucleotides during exercise
deaminase is disrupted by 10 mM phosphate. At concentrations of KC1 optimal for formation of the HMM-adenylate deaminase complex in solution, 90%—95% of the enzyme was readily extracted from myofibrils, while 5%—I 0% remained
was matched by stoichiometric increases in IMP and am-
attached and could not be removed by repeated washes with phosphate-free buffer (3).
study because of the limited sensitivity of the method.
monia. During recovery the removal of IMP matched adenine nucleotide resynthesis. In soleus the small decrease in adenine nucleotides was not matched by a rise in IMP and ammonia,
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the reader is referred to Hintz et al. (32), Saltin and Gollnick (65), and Gauthier (28).
J. M Lowenstein
S42 mt. J. Sports Med. 11(1990)
Other workers have shown that adenylate deaminase and adenylosuccinate synthetase bind to myosin and F-actin, respectively (45, 72). Binding of adenylate deaminase to myosin modifies the regulatory properties of adenylate deaminase and increases its activity. These findings have been
interpreted to suggest that ammonia production during
muscle contraction is based on a close approximation of the two enzymes by the sliding filament mechanism (73). Electrical stimulation of rat hind limb for 30 s leads to a change in the
ratio (adenylate deaminase bound)/(adenylate deaminase free) from about 1/2 to 2/1. In other words, one-half of the enzyme that is free at rest (operationally enzyme that is soluble at low ionic strength) becomes bound during exercise. There was no change in total activity of the enzyme (74).
In contrast, Rahim et al. (54) reported that
seems reasonable to conclude that increases in AMP, ADP, and hydrogen ions are the most important factors that lead to activation of the enzyme, while other effectors, such as GTP and orthophosphate, cause its inhibition or counteract its activation (87).
The kinetic behaviour of adenylate deaminase is modified when the enzyme binds to myosin, heavy meromy-
osin (HMM), or subfragment-2 of HMM. For example, the GTP inhibition is diminished when the purified enzyme is allowed to interact with HMM or subfragment-2 (9). GTP concentration of 1—10 j.tM were used in these experiments. GTP saturation curves in the presence and absence of myosin or my-
osin fragments are not yet available, and it is difficult to extrapolate these observations to the more physiological GTP concentrations of 200—300 IIM.
adenylate deaminase of mouse hind limb is activated by 60%
mM NaF. These findings appear to contradict those of Shiraki et al (74). Adenylate deaminase is very susceptible to proteolytic degradation in vitro. Ranieri-Raggi and Raggi (57) have
suggested that proteolytic activation might occur during strong tetanic contractions. This could possibly explain the observations of Rahin et al. (54), although it does not account for the need to incubate the extracts at 0 C for 30 mm in order to elicit the increase in soluble activity. The contradictory observations probably hinge on the incubation of the muscle extract at 0 C for 30 mm (54), which may allow extensive proteolysis
to occur. If this is the case, then proteolysis would have to occur preferentially in extracts made from exercised muscle as distinct from resting muscle. Regulatory Properties of Adenylate Deaminase
All forms of mammalian adenylate deaminase that have been studied so far are inhibited by GTP (2, 9, 52, 70, 71, 87). All forms are probably also inhibited and activated by
ATP (2, 87). For example, the enzyme from rabbit skeletal muscle becomes increasingly inhibited as the concentration of ATP is increased from 0.005 to 0.1 mM. As the concentration of ATP is increased from 0.1 to 1.0 mM, the inhibition is reversed. As the GTP concentration is raised above 1 mM, the inhibition by GTP is also reversed (87). While the ATP concentration of muscle falls in the range of 6 to 10 mM, that of GTP falls in the range of 0.2 to 0.3 mM (64). This means that the ATP concentration is not, while the GTP concentration is in
The two isozymes of adenylate deaminase which occur in red skeletal muscle show different responses to
ATP, GTP, and orthophosphate. ATP concentrations were kept to 50 tM or less in these studies. As a result, it is difficult to
extrapolate these results to physiological concentrations of ATP (52).
Human Exercise Studies
Exercise studies with human subjects have confirmed experimental observations with rodents. Muscle ammonia increased from 1.3 to 3.6 mol/g dry weight in going from rest to fatigue, respectively. Total adenine nucleotides decreased from 28.7 to 25.1 mol/g dry weight during this time (34). The increase in ammonia of 2.3 was similar to the decrease in total adenine nucleotides of 3.6, but the difference was significant. Glutamine did not change significantly; possibly some ammonia diffused from the muscle. IMP was not determined in this work, but an earlier study showed good agreement between the decrease in total adenine nucleotide and the increase in IMP during exercise (66). The authors calculated that the amount of ammonia released could neutralize only 3 % of the lactic acid formed during exercise. This conclusion is in agreement with that of Dudley and Terjung (19). Katz et al. (34) also concluded that in their experiments the change in pH was of minor importance for the activation of adenylate deaminase during exercise. Additional work with human subjects is described in the next section. Adenylate Deaminase Deficiency
A deficiency of adenylate deaminase in human
the inhibitory range. Physiological concentrations of orthophosphate also inhibit adenylate deaminase. The most important activator of the enzyme is probably ADP, which acts by decreasing the apparent affinity of the enzyme for inhibitors (87, 88). The enzyme has a pH optimum of about 6.2, and this may account in part for the activation of the enzyme during
muscle was first described by Engel et al. (24). Adenylate deaminase activities of gluteus max. were 107.0 and 2.3 tmol/mg nitrogen/I 0 mm at 37 C, in muscles of normal controls and a deficient patient, respectively. Adenylate deaminase activity in the patient's erythrocytes was normal. The
cellular acidosis, which sets in during severe exercise.
patients with this condition had normal levels of the enzyme. It follows that the adenylate deaminase deficiency is not equated
As has already been stated under Functions of the Purine Nucleotide Cycle, the measured contents of ADP and
AMP showed little change between rest, exercise, and recovery from exercise in the perfused hind limb of rat. The cal-
culated contents did change between rest and exercise; the ADP content doubled while that of AMP quadrupled (30). It
patient also suffered from periodic paralysis, but other with periodic paralysis. Five cases of adenylate deaminase deficiency were reported by Fishbein et al. (26); the deficiency was associated with muscular weakness or cramping after exercise. The erythrocyte isozyme of adenylate deaminase was again normal in these patients. Additional cases of adenylate
deaminase deficiency were reported by Shumate et al. (75),
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after 5 s of tetanic stimulation. The increase in activity was confined to the soluble fraction of the hemogenate. The activation was not observed unless the muscle extract was kept at 0 C for 30 mm; it was abolished if the extraction medium contained 20
mt. J. Sports Med. 11 (1990) S43
The Purine Nucleotide Cycle Revised
NH2
-OOcOO—
-OOCCOONH2
O
SAICAR
ON>
Adenytosuccinute lycise
synt hetase
OH OH
OH OH
SAICAR
AICOR
AICAR
—OOc---.-'T—.-cOO-
COO-OOc----.,..1—-
GDP
+Pi Ad e fly I osuc ci flate
Adenylosuccinate synth eta se
OH OH
IMP
lyose OH OH Ad eny I osuccinate
OH OK AMP
Fig. 2 The two reactions catalyzed by adenylosuccinase. The reactions lie on the pathway of purine biosynthesis in the sequence: ribonucleoside AlCOA —+SAICAR —AlCAR .-+5-foramidoimidazole-4-carboxamide 5'-phosphate--+ IMP—+adenylosuccinate-—.AMP. The abbreviations used are: SAICAR, 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleoside 5-phosphate; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside 5-phosphate; AlCOA, 5-aminoimidazole-4-carboxylic acid ribonucleoside 5-phosphate.
who examined 256 muscle biopsies and found that six were deficient in the enzyme. Sabina et a!. (64) made a detailed biochemical study of one patient who possessed < 1 % of the adenylate deaminase activity of 44 controls.
A larger biochemical and functional study involved eight patients with muscle adenylate deaminase deficiency and eleven normal controls (63). Muscle biopsies were obtained at rest and following exercise on a bicycle ergometer to the point of fatigue. Following exercise, controls and deficient patients showed a decrease in ATP of 34% and 6%, respectively; the IMP content of the muscle was 13 times greater
in controls than in deficient patients. Only one deficient patient showed a substantial decrease in ATP following exercise. This patient showed no concomitant increase in IMP. The median decrease of creatine phosphate plus ATP per unit of
work was five times greater in patients deficient in muscle adenylate deaminase. than in controls, which shows that the deficient patients have a lower capacity for energy production than controls. Additional reports of adenylate deaminase deficiency are discussed in reviews by Fishbein (25), van Waarde (86), and Sabina et al. (59).
Adenylate deaminase deficiency is probably an autosomal recessive disease with a carrier frequency of 5 %— 10% (25). The deficiency is associated with skeletal muscle dysfunction, but the clinical picture is variable. Two-thirds of the deficient patients exhibit easy fatigability, post-exercise
muscle cramps, aches, and pains. The AMP formed in the myokinase reaction is not deaminated or is deaminated to a much lesser extent. Some AMP is hydrolyzed to adenosine by
5'-nucleotidase; the adenosine is then deaminated to inosine, which is converted to hypoxanthine. Unlike nucleotides, the nucleosides and base diffuse from the cell. Because of this, measurements of arteriovenous differences are needed in addition to tissue contents to arrive at a proper balance of losses via the 5'-nucelotidase reactions. At present, data on the rate of AMP dephosphorylation are lacking, as are data on the activity of the relevant 5 '-nucleotidase in skeletal muscle. Adenylosuccinate Synthetase
The partially purified enzyme from rat hind limb has Km values for IMP, GTP, and aspartate of 200, 10, and 300 tiM, respectively (30). IMP inhibits the enzyme at concentrations > 260 iiM (76). The maximum concentrations of IMP reached in exercising hind limb of rat was 3.6 mM, which is sufficient to cause a large degree of inhibition of the enzyme in vivo (30). We found that the enzyme is inhibited by GDP, with a Ki of about 10 i.LM (Tornheim and Lowenstein, unpublished). Little is known about changes in GTP and GDP concentration during exercise. Other substances that might act as regulators of the synthetase are discussed by Stayton et a!. (76). Adenylosuccinase
Adenylosuccinase is a bifunctional enzyme in the pathway of purine biosynthesis; it catalyzes the reversible
trans-elimination of fumarate from both adenylosuccinate and SAICAR (Fig. 2). Earlier methods of purifying the enzyme resulted in unstable preparations. A new method yields the pure enzyme in stable form (15). It employs 20%
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OO,1
NH2
GTP
NH2
—
I M Lowenstein
S44 Int.J.SportsMed. 11 (1990)
Inhibition of adenylosuccinase by fluoroanalogs of SAICAR and adenylosuccinate. Values for SAICAR cleavage are approximate because the spectrophotometric assay used in these measurements did not permit accurate readings at substrate concentrations
The Purine Nucleotide Cycle Revised I M Lowenstein Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254, USA
Summary
NH3GDP
+ pi Aspartate + GTP
Key words
adenosine adenylate inonophosphate, deaminase, adenylosuccinase, adenyloscuccinate, adenyloscuccinate lyase, adenylosuccinate synthetase, deamination, exercise, fluoroanalogs, inosine monophosphate, isozymes, phosphofructokinase, phosphorylase, purine nucleotide cycle, reamination
Fig. 1 The purine nucleotide cycle. The reactions shown are catalyzed by a, adenylate deaminase; b, adenylosuccinate synthetase; and c, adenylosuccinate yase (adenylosuccinase).
tate to ammonia and fumarate occurred by a cyclical process
which was termed the purine nucleotide cycle (Fig. 1) (36).
Introduction The sum of reactions 1,2, and 3 yields:
The relationship between muscular work and the production of ammonia was studied in the laboratories of Parnas and Embden in the late 1920s (21—23, 46—48). Both groups recognized that the adenylate deaminase reaction (68) is the major source of ammonia in muscle:
AMP +1120 — IMP + NH3
aspartate + GTP —+ fumarate + NH3 + GDP + P1
(4)
One turn of the cycle results in the deamination of one molecule of aspartate at the expense of the cleavage of one molecule of GTP.
(1)
Reaction I is not reversible under physiological conditions. It was concluded that other reactions must exist for converting IMP back to AMP. These reactions were not identified until 1955 (36):
IMP + asparate + GTP —+ adenylosuccinate + GDP + P(2) (3) adenylosuccinate AMP + fumarate Reactions 2 and 3 are catalyzed by adenylosuccinate synthetase and adenylosuccinate lyase (adenylosuccinase), respectively. In 1971 it was shown that extracts of rat skeletal
muscle can produce ammonia from aspartate under condi-
Subsequently it was shown that the purine nucleotide cycle operates in hind limb of rat during strenuous work. The hind limb was investigated both in vivo and with perfused preparations. Exercise was induced by electrical stimulation. Adenylosuccinate was not present in detectable amounts in resting muscle, but it appeared and rose sharply during exercise to reach a maximum of 21 nmol/g dry weight of muscle. It declined again during recovery from exercise. IMP increased from resting values of about 0.1 .tmol/g dry weight to about 9 mol/g dry weight during strenuous exercise (6 square-wave electrical pulses of 10 ms duration/s) and then declined during recovery. Smaller increases in IMP were observed when the stimulation was decreased to I pulse of 10 ms duration/s, or when resting muscle was perfused with epinephrine, or was made hypoxic. Ammonia increased stoi-
tions that cause the consumption of ATP, i. e., conditions that mimic muscle doing work (38, 80). The conversion of aspar-
chiometrically with IMP (30, 37). These papers also presented measurements of various other metabolites, including certain amino acids, during rest and exercise, as well as calculations of
Int.J. Sports Med. 11(1990) S37—S46
the intracellular concentrations of free ADP and AMP. A
Georgmieme Verlag StuttgartNewYork
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This review is restricted to the operation of the purine cycle in mammalian muscle. A previous review provided a summary of early evidence for the operation of the cycle and of various functions proposed for the cycle. It also provided a brief history of work on ammonia production by muscle and other tissues and of the discovery of the enzymes of the purine nucleotide cycle (36). Recent reviews on the purine nucleotide cycle include those of Terjung et al. (78) and van Waarde (86).
S38 mt. .J. Sports Med. 11(1990)
.1. M Lowenstein
monia as a function of time.
The results obtained with the exercising rat hind limb were interpreted as evidence for the occurrence of the purine nucleotide cycle in skeletal muscle. The following scenario accounts for the experimental observations. During intense exercise, the rate of ATP utilization exceeds the capacity of oxidative phosphorylation and glycolysis to regenerate ATP. As is discussed below, the tendency for this to occur is much greater in muscles with a low than with a high aerobic capacity. The creatine phosphate reservoir acts as a buffer which maintains a high ATP concentration via the creatine kinase reaction: creatine
phosphate + ADP creatine + ATP
(5)
Reaction 5 is assumed to be in equilibrium at rest and during exercise because of the high activity of creatine
kinase found in skeletal muscle. However, NMR measurements with frog gastrocnemius indicate that some deviation from equilibrium may occur during exercise (12). If the capac-
ity of the creatine phosphate/creatine buffer is exceeded, in other words, if most of the creatinephosphate is used up, ADP formed during muscle contraction rises. This leads to an increased formation of AMP via the myokinase reaction:
2 ADP+AMP+ATP
(6)
Reaction 6 is also assumed to be in equilibrium
under all physiological conditions. This assumption is justified by the high activity of myokinase in skeletal muscle, but has not been tested experimentally. Thus, ADP and AMP rise in concert, with AMP rising as the square of the ADP concentration. ADP is the most effective activator of adenylate deaminase prepared from hind limb of rabbit. Moreover, lactic acid accumulates during intense muscular activity. The resulting acidification also serves to activate adenylate deaminase because the enzyme has pH optimum of about 6.2 (87).
Activation of adenylate deaminase leads to the removal of AMP, with the result that reaction 6 is pulled to the right until the ATP/ADP ratio is high:
The functions proposed for the purine nucleotide cycle (36) are here presented in slightly recast form: 1. It serves to regulate the relative concentrations of AMP, ADP, and ATP. In particular it serves to maintain a high ATP/ADP ratio. Insofar as AMP, ADP, and ATP act as substrates, activators, and inhibitors of many reactions, it acts as a general, high level metabolic control mechanism. 2. It is a pathway for the replenishment of citric acid cycle intermediates by generating fumarate from aspartate. 3. It is a pathway for the production of ammonia from amino acids; put differently, it is a pathway
that makes possible the deamination of various amino acids for energy production. Ammonia serves in a small way to diminish the drop in pH caused by lactic acid production. 4. It assists in the regulation of phosphofructokinase activity and hence of glycolysis by regulating the AMP concentration; AMP being an activator of phosphofructokinase. (This is a specific example of the general control mechanism mentioned in 1.) 5. It may serve in the regulation of phosphorylase b by generating IMP during severe exercise while keeping AMP low. Evidence for these proposed functions will now be examined briefly. 1. The maintenance of a high ATP/ADP ratio by the combined action of myokinase and adenylate deaminase cannot be demonstrated by direct metabolite measurements. The ADP and AMP contents of rat hind limb show little or no change between rest, exercise, and recovery from exer-
cise, being 0.60 and 0.11 tmol/g wet weight for ADP and AMP, respectively. Skeletal muscle actin contains one tightly bound molecule of ADP per actin monomer. The binding is so tight that the bound ADP is not available to the pool of free ADP. The content of actin monomer is about 0.6 mol/g wet weight of muscle (41), which is about the same as the total ADP
content. It follows that virtually all of the ADP is bound and unavailable. The standard error of these measurements is too great to use total and bound ADP to calculate free ADP. For this reason, free ADP and AMP were calculated using the assumption that the creatinekinase and myokinase reactions are in equilibrium. The calculated contents of free ADP and AMP in resting hind limb of rat were 19 and 0.12 nmol/g wet weight, respectively, at pH 7.0. After 15 mm of exercise, these rose to 36 and 0.56 nmol/g wet weight, respectively, at pH 6.6. According to these calculations, free ADP accounts for only 3.2%
and 5.4% of the total ADP in muscle at rest and during
(6) (1)
vigorous exercise, respectively. Similarly, free AMP accounts for only 0.11% and 0.50% of the total AMP, respectively. Unlike ADP, there seems to be no protein to which most of the AMP binds tightly. Possibly the AMP is stored in a compart-
(7)
ment where it is not available to myokinase or adenylate deaminase (30). More detailed calculations of adenine nu-
Adenylate deaminase and myokinase are
cleotide contents during rest and exercise were presented by Lowenstein and Goodman (37). The calculated contents of free AMP of 0.12 and 0.56 .tmol/g wet weight correspond to about 0.24 and 1.1 p.M AMP. These concentrations are far below optimum for adenylate deaminase. At 0.1 and 1.0 p.M AMP, adenylate deaminase in rat hind limb can deaminate
AMP +
AlP
AMP + H20 — IMP +
NH3
2ADP+ H20 —+ ATP +
IMP +
2ADP Sum:
Functions of the Purine Nucleotide Cycle
NH3
among the most active enzymes of skeletal muscle and their combined action explains the formation of IMP during severe exercise. IMP accumulates because adenylate deaminase occurs in far higher activity than adenylosuccinate synthetase and adenylosuccinase; in other words, the reactions that convert IMP back to AMP cannot keep up with the rate of IMP formation. The question of whether or not the reactions of the purine nucleotide cycle operate concurrently is dealt with below.
AMP at maximum rates of 0.02 and 0.2 p.mol/g wet weight/mm, respectively. These rates are substantially greater than the observed rates of IMP formation. This implies that adenylate deaminase is not fully activated under these conditions (30).
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detailed discussion of these data is outside the scope of this review. The stoichiometric increase in ammonia and IMP indicates that ammonia diffuses only slowly from muscle. A similar conclusion was reached by Rosado et al. (58), who injected rats with NH4C1 and studied the resulting distribution of am-
The proposed function of maintaining a high ATP/ADP ratio is therefore supported by calculations of metabolite contents. Verification independent of such calculations is highly desirable. This would probably require determination of adenine nucleotide and other metabolite contents in different compartments of the muscle cell; techniques for carlying out such measurements are becoming available. 2. The contents of citric acid cycle intermediates were measured in rat hind limb during rest, exercise, and recovery from exercise. The contents of fumarate and malate increased four-fold and the sum of the contents of all citric acid cycle intermediates doubled after 10 mm of exercise. All metabolites returned to resting values during recovery from exercise. These observations are consistent with the proposal that the purine nucleotide cycle serves to rep1enish citric acid cycle intermediates. Hadacidin (N-formylhydroxyaminoacetate) is a competitive inhibitor with respect to aspartate in the adenylosuccinate synthetase reaction. Accumulation of IMP in rat
hind limb during 10 mm of exercise was 1.5 and 2.5 mol/g dry weight in the absence and presence of hadacidin, respectively. The difference of 1.0 imol represents the partial inhibi-
tion of adenylosuccinate synthetase by hadacidin. The increase in fumarate plus malate over the same interval was 1.14 and 0.54 .tmo1/g dry weight, respectively. Thus, hadacidin inhibited fumarate plus malate production by 53%. Hadacidin has no effect on the levels of IMP, fumarate, and malate in unexercised controls. These experiments constitute strong evidence that part, if not all, of the increase in citric acid cycle intermediates observed during exercise is due to the operation of
the purine nucleotide cycle. Other pathways for increasing citric acid cycle intermediates do not seem to operate in skeletal muscle (4, 5). Terjung et al. (78) believe that these data cannot be interpreted to show that operation of the purine nucleotide cycle serves to increase citric acid cycle intermediates in contracting muscle because the hind limbs used by us were composed of FG and FOG fibers. They fail to provide an alternative explanation for the higher accumulation of IMP and for
the inhibition of fumarate plus malate production in the presence of hadacidin which we observed during the operation of the cycle, regardless of whether the fibers were mixed or not. They also fail to provide alternative interpretations for the accumulation of malate and fumarate during the operation of the cycle in the absence of hadacidin. In mixed muscle, for a given total activity, if one fiber type shows low activity of the purine nucleotide cycle, then the other fiber type must show a correspondingly higher activity. Oddly, in their own paper they conclude that increased IMP accumulation in the presence of hadacidin in mixed muscle is strong evidence that turnover of the purine nucleotide cycle occurred (44). These authors showed that FG fibers of plantaris (50 %—55 % FG, 40%—45 % FOG) were the major site of purine nucleotide cycle activity.
AlCAriboside has been used to partially disrupt the purine nucleotide cycle at the level of adenylosucci-
nate synthetase and adenylosuccinase, while leaving the adenylate deaminase reaction to function (27). Treatment with AlCAriboside results in a pronounced decrease in tension both with single, non-tetanizing pulses and with tetanic stimulation (a detailed explanation of the mode of action of this compound is given under Adenylosuccinas). During both aerobic and anaerobic stimulation of gastrocnemius, adeny-
mt. J. Sports Med 11(1990) S39 late deaminase in AlCAriboside-treated animals was more active and adenylosuccinate synthetase and adenylosuccinase were less active than in controls. Total energy production was impaired in the test group, but lactate production was similar
in test and control groups. The authors concluded that the purine nucleotide cycle plays an anaplerotic role in providing citric acid cycle intermediates that enhance energy production in contracting skeletal muscle. Total citric acid cycle intermediates were not determined in this study, but changes in malate
and citrate in response to exercise were similar in salinetreated and AlCAriboside-treated animals. The decrease in tension may therefore be due to other effects of AlCAriboside or its metabolic products (see Adenylosuccinase).
The original demonstration of the operation of the purine nucleotide cycle was performed with a soluble fraction obtained from skeletal muscle (cytosol) (38, 80). Skeletal
muscle cytosol and mitochondria were prepared separately and combined to demonstrate that the generation of fumarate by the purine nucleotide cycle in the cytosol can serve to accelerate the oxidation of pyruvate via the citric acid cycle in the mitochondria. In this system, the acceleration of pyruvate oxidation depended on the addition of GTP, aspartate, and AMP or IMP (69).
The functioning of the purine nucleotide cycle was examined further in particle-free extracts of gastrocnemius and soleus. The activities of the enzymes of the cycle increased in parallel as the ATP/ADP ratio was lowered from 50 to I, i. e., a low energy state leads to an activation of the operation of the cycle. Interestingly, the inhibition of adenylosuccinate synthetase by high concentrations of IMP (see below) was
not observed by these workers. Adenylosuccinate (0.6 mM) activated adenylate deaminase two-fold (39). The adenylosuccinate concentration of 0.6 mM is much higher than the maximum reported in skeletal muscle. It is therefore unlikely that adenylosuccinate is a physiological activator of adenylate deaminase.
3. Ammonia production during muscle contraction has been demonstrated repeatedly (7, 34, 90, to mention only a few recent papers on this topic involving human subjects). The best evidence that the ammonia arises via the deamination of AMP is the observed stoichiometry between IMP and ammonia production. Evidence that glutamate dehydrogenase is not involved in ammonia production by exercising muscle rests on a comparison of its activity in the direction of deamination with the observed rates of ammonia production (30, 37). The best evidence of the involvement of the purine nucleotide cycle in the deamination of amino acids in skeletal muscle was obtained with 15N-labelled leucine. Depending on when it was provided to the exercised, perfused hind limb, between 14% and 24% of the amino nitrogen used in the reamination of IMP originated from the 15N-labelled amino acid (31). 4. The regulatory properties of phosphofructokinase are complex. The enzyme is inhibited by physiological concentrations of ATP and by citrate when ATP is in the inhibitory range. The ATP inhibition is relieved by its degradative products, namely orthophosphate, ADP, and AMP. Other
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The Purine Nucleotide Cycle Revised
J. M Lowenstein
S40 mt. I Sports Med. 11(1990)
bition, are fructose-6-phosphate (F6P), fructose-i ,6-diphosphate (Fl ,6diP), glucose-i ,6-diphosphate (G 1 ,6diP), cAMP (83), and fructose-2,6-diphosphate (F2,6diP). The activation by Fl,6diP is enhanced synergistically by AMP (81). F2,6diP was not available in 1976; it was shown subsequently that synergism also occurs between F2,6diP and AMP (84, 85). ADP is a weak activator, but its effect is increased greatly by
orthophosphate (49). The AlP inhibition increases with decreasing pH. For example, with 5 mM ATP, the Kapp for F6P is increased by a factor of 50 as the pH is lowered from 7.7
to 6.8. In other words, lowering the pH diminishes the apparent affinity of the enzyme for F6P. This effect is abolished
by 10 .tM F2,6diP. Other effectors are not as powerful as F2,6diP, but combinations of AMP and Gl,6diP are as effective as F2,6diP (18). Most studies of the regulatory behaviour of phosphofructokinase were conducted at enzyme concentrations well below those which occur in the muscle cell. When the enzyme is used at physiological concentrations, or in the presence of ethylene glycol, which favors aggregation of the dilute enzyme, the ATP inhibition is greatly diminished. However, activating effectors, such as AMP and orthophosphate, still work by increasing the apparent affinity of the enzyme for F6P (10). F2,6diP is the most powerful activator of phosphofructokinase. Its concentration in resting skeletal muscle is very low and actually decreases during exercise. It is therefore difficult to assign a regulatory role for F2,6diP in glycolysis during muscle contraction, although it may have such a role in resting muscle in response to epinephrine (33). Studies of the oscillatory behaviour of glycolysis in cell-free extracts of rat skeletal muscle show that F2,6diP is not involved in the control of the oscillations. If 0.5 .tM F2,6diP is added to the extracts,
the oscillations are abolished, but 0.1 .LM F2,6diP does not prevent the oscillations. In the presence of physiological concentrations of ATP and citrate, physiological concentrations of F2,6diP do not block the oscillations, but increase their
frequency. The physiological role of F2,6diP in skeletal muscle remains uncertain. Its concentration in vivo is only about 8% of that of phosphofructokinase subunits. Moreover, appropriate changes in F2,6diP have not been observed upon initiation of muscular exercise (79).
5. Activation of phosphorylase b by IMP and AMP was first reported by Con et al. (17), but a physiological role for IMP was ruled out because IMP is a much weaker activator than AMP. Activation of the enzyme by IMP was suggested as a mechanism that could account for the high rates of glycolysis observed in mice deficient in phosphorylase kinase (ICR/IAn mice). AMP changes little during exercise in such mice, but IMP rises more rapidly and to higher levels than in normal mice. These observations were interpreted as showing that IMP can act as an activator of phosphorylase b not only in muscle of ICR/IAn mice, but also in muscle of normal mice
was below control levels (6). IMP accumulated rapidly in exercising muscle, reaching 1,200 .tM after 10 s, while the (calcu-
lated) AMP concentration was around i tM. At this point, maximum conversion to the a form of phosphorylase had occurred. The accumulated IMP was sufficient to activate the a form substantially. After 20 s of exercise, IMP had reached 1560 .LM and AMP around 4 .tM. This IMP concentration was sufficient to activate phosphorylase b about six-fold over the basal activity, whereas the AMP concentration was insufficient to activate the enzyme. A six-fold activation of the b form was not sufficient to account for the observed rates of glycolysis; it was speculated that other activators may occur in muscle which may act in concert with IMP (6). Rahim et al. (56) observed a 40% decline in phosphorylase a in mouse hind limb between 5 and 60 s of exercise. Reversal of the phosphorylase b
to a conversion, despite continued contractile activity, was also studied by Conlee et al. (16). These workers found that the
conversion was transient in both plantaris (41 % FG, 53% FOG, and 6% SO) and soleus (15% FOG and 85% SO). In summary, kinetic studies with muscle extracts and the purified enzyme, as well as measurements of changes in IMP contents
during exercise, show that IMP can act as an activator of phosphorylase b. Under these conditions the AMP concentration is too low to be effective as an activator. Activation of the b
form by IMP does not appear to be sufficient to account completely for the observed rates of glycogenolysis, but IMP may act in concert with other, unidentified activators. Concurrent Versus Segmented Operation of the Cycle
As has already been mentioned, IMP accumulates during vigorous exercise because adenylate deaminase (reaction i) operates at higher rates than adenylosuccinate synthetase (reaction 2) and adenylosuccinase (reaction 3); in other words, the reactions that convert IMP back to AMP cannot keep up with the rate of IMP formation. The question has been raised whether reactions 2 and 3 operate at all during the phase when IMP is being formed by reaction 1 (43, 44, 78). Hadacidin inhibits adenylosuccinate synthetase (see above). In the exercising hind limb, a greater accumulation of IMP occurred in the presence of hadacidin than in its absence within 1 mm after the onset of exercise and thereafter. Maximum accumulation on IMP occurred 10 mm after starting the exercise. It follows that reaction 2 must have been functioning long before maximum accumulation of IMP had occurred (5). Other evidence showed that adenylosuccinate rises and reaches near maximum levels during exercise (30, 37). It was concluded that the cycle operates even while AMP is being deaminated. It is not known at which point during or after exercise reactions 2 and 3 reach their maximum rates. It seems likely that this will depend on the type of muscle being studied. Red Versus White Muscle
(55, 56).
Comparison of the activation of phosphorylase b by AMP and IMP in vitro showed that it was necessary to use 50 times as much IMP in order to obtain the same degree of activation as is obtained with AMP (6). Assays of the activities
of phosphorylase a and b during exercise in hind limb showed that the conversion of the b to the a form reached a maximum
within 10 s after the start of exercise. However, after 20 s phosphorylase a had declined considerably, and by 1 mm it
Skeletal muscle fibers have been classified into three types on the basis of their biochemical and physiological properties, namely fast-twitch-glycolytic (FG), fast-twitch-oxidative-glycolytic (FOG), and slow-twitch-oxidative (SO) (8, 50). Earlier terminologies for different fiber types were compared by Peter et al. (50). For a detailed discussion of skeletal muscle fiber types, their function, and their enzyme profiles,
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activators, all of which appear to act by relieving the ATP inhi-
mt. J. Sports Med. 11 (1990) S41
The Purine Nucleotide Cycle Revised
Different skeletal muscle fiber types contain different activities of adenylate deaminase. Raggi et al. (53) showed that white muscles of rabbit had much higher adenylate deaminase, myokinase, and creatine kinase activities than red muscles. These authors also examined red and white muscles of rat and pigeon, with similar results, Gerez and Kir-
even though more intense stimulation was employed. Dudley and Terjung (19) developed these findings further. They re-
ported that upon moderately intense electrical stimulation, FG muscle fibers ("white" fibers) showed a decrease in total adenine nucleotides by as much as 50%, depletion of their creatine phosphate, and severe acidosis. Under the same conditions, the FOG fibers of the same muscle ("red" fibers) showed little depletion of adenine nucleotides, only a partial decrease in creatine phosphate, and much less acidosis. Elimi-
sten (29) had shown previously that muscles poor in mitochondna, such as leg muscle of rat, produce more ammonia during exercise than do muscles rich in mitochondria, such as breast
nation of the blood flow before initiating the contractions abolished the distinction between the FG and FOG fibers.
muscle of pigeon. Differences in adenylate deaminase and
chiometric loss of ATP. Dudley and Terjung (20) concluded that cellular acidosis is a major factor in causing activation of adenylate deaminase, especially in FG fibers. Whitlock and
adenylosuccinate lyase activities of different fiber types of rat
skeletal muscles were documented by Winder et al. (91). Adenylate deaminase activity was highest in white quadriceps
(mainly FG), next highest in red quadriceps (mainly FOG), and lowest in soleus (SO). The activities were in the ratio 100:84:40, respectively. It should be pointed out that in each muscle adenylate deaminase activity measured under conditions of Vmax is present in great excess over the deaminase ac-
tivity actually expressed in intact muscle. In other words, adenylate deaminase in vivo operates at only a small fraction of its Vmax. A significant correlation was observed between
adenylate deaminase and phosphofructokinase activities (r=0.97).
Both now produced a high amount of IMP and a stoi-
Terjung (89) showed subsequently that during prolonged ischemic stimulation even soleus (SO) undergoes a large depletion in adenine nucleotides, which can be accounted for
by the accumulation of IMP. The authors believe that the limited depletion of the adenine nucleotide pool that is observed during exercise in muscles such as soleus is due in part to the limited accumulation of lactic acid, because when conditions are used which do lead to a large accumulation of lactic acid, such as prolonged stimulation during ischemia, a large decrease in ATP and a stoichiometric increase in IMP does occur.
The abundance of adenylate deaminase
Adenylate Deaminase Structure and Expression
mRNA in different skeletal muscle fiber types of rat was assessed by Sabina et al. (61). The mRNA was most abundant in outer gastrocnemius (FG), less abundant in inner gastrocne-
The complementary DNA (cDNA) of adenylate deaminase messenger RNA (mRNA) has been cloned,
mius (FOG), and least abundant in soleus (SO), and paral-
and the amino acid sequence of the enzyme deduced (61). The
lelled the differences in adenylate deaminase activity found by Raggi et al. (53) and Winder et al. (91).
enzyme has a molecular weight of about 80,000. Three
Treadmill training for 12 weeks resulted in a 29% decline in adenylate deaminase activity in red quadriceps, but resulted in no change in the other types of muscle. Adenylosuccinase activity was highest in red quadriceps and approximately equal in white quadriceps and soleus. Its activity remained unchanged during the exercise regimen (91).
Cat biceps brachii (>75% FG) and soleus
(>92% SO) were analyzed by NMR. The creatine phosphate/creatine ratios calculated from the data so ob-
isoforms of adenylate deaminase have been identified during skeletal muscle development. An embryonic isoform appears in the hind limb of rat between 7 and 14 days of gestation; this form is also expressed in non-muscle tissues in the perinatal and adult rat. A second form appears 4—6 days before birth and persists for 2—3 weeks after birth; this form is expressed only in
skeletal muscle. The adult isoform appears after birth, and reaches maximum levels after 3 weeks; this form, too, is expressed only in skeletal muscle (40). The expression of these three, stage-specific transcripts during development has been studied further in myocytes in culture and in hind limb of rat (62).
tained were 175 in the FG and 2.1 in the SO muscles. The calcu-
lated ADP concentration in FG was 0.3 tM and in SO was 14 1XM. Intracellular orthophosphate was 3 mM in FG and 10
mM in SO. The intracellular pH was 7.0 in both types of muscles (42). Similar measurements were presented for rat hind limb, except that in this study the limb was subjected to exercise (35). IMP formation could not be quantitated in this
Intracellular Localization of Adenylate Deaminase
Adenylate deaminase has been shown to be at-
tached to the A band of the sarcomere (3). In solution, the
Meyer and Terjung (43) showed that during
muscle enzyme forms a tight complex with heavy meromyosin and with subfragment-2 of heavy meromyosin. Subfragment-2 is believed to be involved in the interaction of myosin and actin (1). The complex formed between pure HMM and adenylate
tetanic stimulation of rat muscles in situ, the total adenine nucleotide content of gastrocnemius (FG and FOG) fell by as much as 50%, whereas that of soleus (SO) fell by only 10%. In gastrocnemius the fall in adenine nucleotides during exercise
deaminase is disrupted by 10 mM phosphate. At concentrations of KC1 optimal for formation of the HMM-adenylate deaminase complex in solution, 90%—95% of the enzyme was readily extracted from myofibrils, while 5%—I 0% remained
was matched by stoichiometric increases in IMP and am-
attached and could not be removed by repeated washes with phosphate-free buffer (3).
study because of the limited sensitivity of the method.
monia. During recovery the removal of IMP matched adenine nucleotide resynthesis. In soleus the small decrease in adenine nucleotides was not matched by a rise in IMP and ammonia,
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the reader is referred to Hintz et al. (32), Saltin and Gollnick (65), and Gauthier (28).
J. M Lowenstein
S42 mt. J. Sports Med. 11(1990)
Other workers have shown that adenylate deaminase and adenylosuccinate synthetase bind to myosin and F-actin, respectively (45, 72). Binding of adenylate deaminase to myosin modifies the regulatory properties of adenylate deaminase and increases its activity. These findings have been
interpreted to suggest that ammonia production during
muscle contraction is based on a close approximation of the two enzymes by the sliding filament mechanism (73). Electrical stimulation of rat hind limb for 30 s leads to a change in the
ratio (adenylate deaminase bound)/(adenylate deaminase free) from about 1/2 to 2/1. In other words, one-half of the enzyme that is free at rest (operationally enzyme that is soluble at low ionic strength) becomes bound during exercise. There was no change in total activity of the enzyme (74).
In contrast, Rahim et al. (54) reported that
seems reasonable to conclude that increases in AMP, ADP, and hydrogen ions are the most important factors that lead to activation of the enzyme, while other effectors, such as GTP and orthophosphate, cause its inhibition or counteract its activation (87).
The kinetic behaviour of adenylate deaminase is modified when the enzyme binds to myosin, heavy meromy-
osin (HMM), or subfragment-2 of HMM. For example, the GTP inhibition is diminished when the purified enzyme is allowed to interact with HMM or subfragment-2 (9). GTP concentration of 1—10 j.tM were used in these experiments. GTP saturation curves in the presence and absence of myosin or my-
osin fragments are not yet available, and it is difficult to extrapolate these observations to the more physiological GTP concentrations of 200—300 IIM.
adenylate deaminase of mouse hind limb is activated by 60%
mM NaF. These findings appear to contradict those of Shiraki et al (74). Adenylate deaminase is very susceptible to proteolytic degradation in vitro. Ranieri-Raggi and Raggi (57) have
suggested that proteolytic activation might occur during strong tetanic contractions. This could possibly explain the observations of Rahin et al. (54), although it does not account for the need to incubate the extracts at 0 C for 30 mm in order to elicit the increase in soluble activity. The contradictory observations probably hinge on the incubation of the muscle extract at 0 C for 30 mm (54), which may allow extensive proteolysis
to occur. If this is the case, then proteolysis would have to occur preferentially in extracts made from exercised muscle as distinct from resting muscle. Regulatory Properties of Adenylate Deaminase
All forms of mammalian adenylate deaminase that have been studied so far are inhibited by GTP (2, 9, 52, 70, 71, 87). All forms are probably also inhibited and activated by
ATP (2, 87). For example, the enzyme from rabbit skeletal muscle becomes increasingly inhibited as the concentration of ATP is increased from 0.005 to 0.1 mM. As the concentration of ATP is increased from 0.1 to 1.0 mM, the inhibition is reversed. As the GTP concentration is raised above 1 mM, the inhibition by GTP is also reversed (87). While the ATP concentration of muscle falls in the range of 6 to 10 mM, that of GTP falls in the range of 0.2 to 0.3 mM (64). This means that the ATP concentration is not, while the GTP concentration is in
The two isozymes of adenylate deaminase which occur in red skeletal muscle show different responses to
ATP, GTP, and orthophosphate. ATP concentrations were kept to 50 tM or less in these studies. As a result, it is difficult to
extrapolate these results to physiological concentrations of ATP (52).
Human Exercise Studies
Exercise studies with human subjects have confirmed experimental observations with rodents. Muscle ammonia increased from 1.3 to 3.6 mol/g dry weight in going from rest to fatigue, respectively. Total adenine nucleotides decreased from 28.7 to 25.1 mol/g dry weight during this time (34). The increase in ammonia of 2.3 was similar to the decrease in total adenine nucleotides of 3.6, but the difference was significant. Glutamine did not change significantly; possibly some ammonia diffused from the muscle. IMP was not determined in this work, but an earlier study showed good agreement between the decrease in total adenine nucleotide and the increase in IMP during exercise (66). The authors calculated that the amount of ammonia released could neutralize only 3 % of the lactic acid formed during exercise. This conclusion is in agreement with that of Dudley and Terjung (19). Katz et al. (34) also concluded that in their experiments the change in pH was of minor importance for the activation of adenylate deaminase during exercise. Additional work with human subjects is described in the next section. Adenylate Deaminase Deficiency
A deficiency of adenylate deaminase in human
the inhibitory range. Physiological concentrations of orthophosphate also inhibit adenylate deaminase. The most important activator of the enzyme is probably ADP, which acts by decreasing the apparent affinity of the enzyme for inhibitors (87, 88). The enzyme has a pH optimum of about 6.2, and this may account in part for the activation of the enzyme during
muscle was first described by Engel et al. (24). Adenylate deaminase activities of gluteus max. were 107.0 and 2.3 tmol/mg nitrogen/I 0 mm at 37 C, in muscles of normal controls and a deficient patient, respectively. Adenylate deaminase activity in the patient's erythrocytes was normal. The
cellular acidosis, which sets in during severe exercise.
patients with this condition had normal levels of the enzyme. It follows that the adenylate deaminase deficiency is not equated
As has already been stated under Functions of the Purine Nucleotide Cycle, the measured contents of ADP and
AMP showed little change between rest, exercise, and recovery from exercise in the perfused hind limb of rat. The cal-
culated contents did change between rest and exercise; the ADP content doubled while that of AMP quadrupled (30). It
patient also suffered from periodic paralysis, but other with periodic paralysis. Five cases of adenylate deaminase deficiency were reported by Fishbein et al. (26); the deficiency was associated with muscular weakness or cramping after exercise. The erythrocyte isozyme of adenylate deaminase was again normal in these patients. Additional cases of adenylate
deaminase deficiency were reported by Shumate et al. (75),
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after 5 s of tetanic stimulation. The increase in activity was confined to the soluble fraction of the hemogenate. The activation was not observed unless the muscle extract was kept at 0 C for 30 mm; it was abolished if the extraction medium contained 20
mt. J. Sports Med. 11 (1990) S43
The Purine Nucleotide Cycle Revised
NH2
-OOcOO—
-OOCCOONH2
O
SAICAR
ON>
Adenytosuccinute lycise
synt hetase
OH OH
OH OH
SAICAR
AICOR
AICAR
—OOc---.-'T—.-cOO-
COO-OOc----.,..1—-
GDP
+Pi Ad e fly I osuc ci flate
Adenylosuccinate synth eta se
OH OH
IMP
lyose OH OH Ad eny I osuccinate
OH OK AMP
Fig. 2 The two reactions catalyzed by adenylosuccinase. The reactions lie on the pathway of purine biosynthesis in the sequence: ribonucleoside AlCOA —+SAICAR —AlCAR .-+5-foramidoimidazole-4-carboxamide 5'-phosphate--+ IMP—+adenylosuccinate-—.AMP. The abbreviations used are: SAICAR, 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleoside 5-phosphate; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside 5-phosphate; AlCOA, 5-aminoimidazole-4-carboxylic acid ribonucleoside 5-phosphate.
who examined 256 muscle biopsies and found that six were deficient in the enzyme. Sabina et a!. (64) made a detailed biochemical study of one patient who possessed < 1 % of the adenylate deaminase activity of 44 controls.
A larger biochemical and functional study involved eight patients with muscle adenylate deaminase deficiency and eleven normal controls (63). Muscle biopsies were obtained at rest and following exercise on a bicycle ergometer to the point of fatigue. Following exercise, controls and deficient patients showed a decrease in ATP of 34% and 6%, respectively; the IMP content of the muscle was 13 times greater
in controls than in deficient patients. Only one deficient patient showed a substantial decrease in ATP following exercise. This patient showed no concomitant increase in IMP. The median decrease of creatine phosphate plus ATP per unit of
work was five times greater in patients deficient in muscle adenylate deaminase. than in controls, which shows that the deficient patients have a lower capacity for energy production than controls. Additional reports of adenylate deaminase deficiency are discussed in reviews by Fishbein (25), van Waarde (86), and Sabina et al. (59).
Adenylate deaminase deficiency is probably an autosomal recessive disease with a carrier frequency of 5 %— 10% (25). The deficiency is associated with skeletal muscle dysfunction, but the clinical picture is variable. Two-thirds of the deficient patients exhibit easy fatigability, post-exercise
muscle cramps, aches, and pains. The AMP formed in the myokinase reaction is not deaminated or is deaminated to a much lesser extent. Some AMP is hydrolyzed to adenosine by
5'-nucleotidase; the adenosine is then deaminated to inosine, which is converted to hypoxanthine. Unlike nucleotides, the nucleosides and base diffuse from the cell. Because of this, measurements of arteriovenous differences are needed in addition to tissue contents to arrive at a proper balance of losses via the 5'-nucelotidase reactions. At present, data on the rate of AMP dephosphorylation are lacking, as are data on the activity of the relevant 5 '-nucleotidase in skeletal muscle. Adenylosuccinate Synthetase
The partially purified enzyme from rat hind limb has Km values for IMP, GTP, and aspartate of 200, 10, and 300 tiM, respectively (30). IMP inhibits the enzyme at concentrations > 260 iiM (76). The maximum concentrations of IMP reached in exercising hind limb of rat was 3.6 mM, which is sufficient to cause a large degree of inhibition of the enzyme in vivo (30). We found that the enzyme is inhibited by GDP, with a Ki of about 10 i.LM (Tornheim and Lowenstein, unpublished). Little is known about changes in GTP and GDP concentration during exercise. Other substances that might act as regulators of the synthetase are discussed by Stayton et a!. (76). Adenylosuccinase
Adenylosuccinase is a bifunctional enzyme in the pathway of purine biosynthesis; it catalyzes the reversible
trans-elimination of fumarate from both adenylosuccinate and SAICAR (Fig. 2). Earlier methods of purifying the enzyme resulted in unstable preparations. A new method yields the pure enzyme in stable form (15). It employs 20%
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OO,1
NH2
GTP
NH2
—
I M Lowenstein
S44 Int.J.SportsMed. 11 (1990)
Inhibition of adenylosuccinase by fluoroanalogs of SAICAR and adenylosuccinate. Values for SAICAR cleavage are approximate because the spectrophotometric assay used in these measurements did not permit accurate readings at substrate concentrations
