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University of Groningen
Operation of the purine nucleotide cycle in animal tissues.
van Waarde, Aren
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Biological Reviews
DOI:
10.1111/j.1469-185X.1988.tb00632.x
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van Waarde, A. (1988). Operation of the purine nucleotide cycle in animal tissues. Biological Reviews,
63(2), 259-298. DOI: 10.1111/j.1469-185X.1988.tb00632.x
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259
BIol. Rev. (1988), 63,pp. 259-298
Printed in Great Britain
OPERATION OF T H E PURINE NUCLEOTIDE CYCLE I N
ANIMAL TISSUES
BY AREN VAN WAARDE
Department of Animal Physiology, Gorlaeus Laboratories, University of Leiden,
Einsteinweg 5 , P.O. Box 9502, 2300 RA Leiden, The Netherlands
(Received
22 July I 987,
accepted 4 January I 988)
CONTENTS
I. Introduction .
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( I ) Discovery of the component reactions of the cycle
(2) Early evidence for ‘purine nucleotide cycling’ .
(3) Proposed physiological functions of the cycle .
11. Liver
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111. Kidney .
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IV. Platelets .
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V. Brain
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VI. Vertebrate skeletal muscle
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VII. Invertebrate tissues.
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VIII. Conclusion
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IX. Summary
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X. Acknowledgements .
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XI. References
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XII. Addendum
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26.5
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I. INTRODUCTION
T h e purine nucleotide cycle (PNC) is a group of three reactions involved in the
reversible deamination of adenosine 5’-monophosphate (AMP). Deamination is
catalysed by the enzyme adenylate deaminase (E.C. 3.5.4.6)and it results in the
formation of IMP (reaction I ) :
AMP + H,O
+ IMP
+ NH,.
(1)
T h e regeneration of AMP from IMP occurs in two steps (reactions 2 and 3), which are
catalysed by adenylosuccinate synthetase (E.C. 6.3.4.4)and adenylosuccinate lyase
(E.C. 4.3.2.2):
aspartate
+ GTP + IMP + adenylosuccinate + GDP + Pi,
adenylosuccinate +AMP + fumarate.
(2)
(3)
T h e net reaction of the cycle is therefore
aspartate
+ GTP + H,O + fumarate + GDP + Pi + NH,.
(4)
A description of the discovery of the cycle will be followed by a discussion of its
operation in various tissues of vertebrates.
260
ARENVAN WAARDE
( I ) Discovery of the component reactions of the cycle
In 1927, Parnas & Mozolowski described how homogenization of skeletal muscle of
vertebrates in 0.9 yo NaCl results in a rapid ( < 90 s), anaerobic production of ammonia.
Ammoniagenesis is greater in minced white muscle than in red muscle, and it also takes
place in intact muscle upon electrical stimulation. Parnas et al. ( I 927) subsequently
measured ammonia levels in blood drawn from the human cubital vein during rest and
during muscular exercise. Blood ammonia proved to be raised three- to fourfold by
exercise. Ammonia produced during muscular work therefore seems to be lost to the
circulation.
Embden et ad. (1928) demonstrated that ammonia production in homogenates of frog
and rabbit muscle is not increased by addition of exogenous urea, but strongly
stimulated upon addition of AMP, suggesting AMP to be the ammoniogenic substrate.
Embden & Wassermeyer (1928) showed that the muscle extract converts AMP to IMP,
so that only one-fifth of the total purine nitrogen is liberated as ammonia.
Schmidt (1928) examined deamination processes in a homogenate from rabbit
muscle. Whereas A M P and adenosine were rapidly broken down, adenine was hardly
deaminated. AMP-deaminase and adenosine deaminase were found to be different
proteins. A partially purified preparation of AMP-deaminase showed a pH-optimum of
about 6 . Although AMP was very rapidly converted to IMP, A T P proved to be
unsuitable as a substrate.
Parnas ( 1 9 2 9 a, b ) developed techniques for the measurement of small amounts of
purine nucleotides. Thus, he was able to determine that the ammonia formed in
anaerobically stimulated frog muscle is equivalent to the net amount of adenine
nucleotide converted to IMP. In the presence of oxygen, however, ammonia formation
was much greater than the decline of adenylates or the increase of the IMP-level. Parnas
explained this by the farsighted assumption that A M P can be resynthesized from I M P
under aerobic conditions, using an amino donor other than ammonia. He even
proposed amino acids as the most likely donors of the amino group.
After the discovery of adenylate deaminase by Schmidt in 1928, it lasted until 1955
before the mechanism of the aerobic reamination was elucidated. Abrams 8z Bentley
(1955) studied the process in a high-speed supernatant of rabbit bone marrow. They
demonstrated aspartate to be a good amino donor, whereas an energy-rich phosphate
compound seemed to be required. Carter & Cohen ( I 955) partially purified the enzyme
from yeast which catalyses reaction (3). T h e authors rightly assumed adenylosuccinate
to be an intermediate in the mechanism for incorporation of the amino group in adenine
nucleotides. Subsequently, Lieberman ( I 956) purified a protein from Escherichia coli
which synthesizes adenylosuccinate according to mechanism 2. He showed the enzyme
to be specific for GTP as the energy source and for aspartate as the amino donor.
Reactions 2 and 3 were later observed in vertebrate skeletal muscle (Newton & Perry,
1957, 1960; Davey, 1961)and vertebrate liver (Davey, 1961). With the emergence of
more sensitive techniques of detection, they have been shown to be ubiquitous in all
organisms (from bacteria to man) and in all tissues which are capable of de novo purine
synthesis (Lowenstein, 1972).
The purine nucleotide cycle
(2)
26 I
Early evidence for 'purine nucleotide cycling
Although a forward and backward mechanism for the conversion of A M P into I M P
has been shown to exist, this does not prove that the enzymes AMP-deaminase,
adenylosuccinate synthetase and adenylosuccinase operate as a cycle. In theory, it is
quite possible for the deamination and reamination pathways to function independently
in the catabolism and anabolism of purine nucleotides. If A M P formed by
adenylosuccinate synthetase is rapidly converted to ATP, and I M P formed by AMPdeaminase is immediately dephosphorylated, no ' cycling ' will occur. T h e turnover rate
of the 6-amino-group of adenine nucleotides will in that case be equal to the turnover
of the ring nitrogens. If on the other hand purine skeletons pass several amination/
deamination sequences before being ultimately catabolized, purine nucleotides will
serve a catalytic function in the liberation of ammonia from aspartate and the turnover
of the 6-NH2 group will be much greater than turnover of nitrogen in the purine
ring.
By feeding rats ['5N]ammonium citrate, Barnes & Schoenheimer ( I 943) demonstrated that 15N incorporated in nucleic acids of rat liver is fairly equally distributed
between the amino-N and the purine ring nitrogens. Liver AMP-deaminase therefore
seems to operate more or less independently of the IMP-reamination pathway and there
appears to be little purine nucleotide cycling.
In similar feeding experiments, however, Kalckar & Rittenberg (r947) showed that
muscle incorporates the label predominantly into the 6-NH2-group. After 7-8 hours,
the amino group was labelled 50 times more strongly than the nitrogens of the purine
ring. In muscle, turnover of the amino group is thus much higher than that of the ring
nitrogens, indicating the occurrence of active purine nucleotide cycling. Kalckar &
Rittenberg's data were later confirmed by Newton & Perry (1957, 1960).
Evidence for a cyclical operation of muscle AMP-deaminase, adenylosuccinate
synthetase and adenylosuccinate lyase has also been presented by Wajzer et a l . (1956).
These authors mounted frog skeletal muscle fibres in the beam of a UV spectrophotometer. Absorption was monitored at different wavelengths, while the fibre
was stimulated. A rapid decrease at 265 nm was accompanied by an increase at 240 nm
and a stable absorption at 248 nm. After a single contraction, absorption at 240 and
265 nm returned slowly to the control values. This observation suggests that reversible
degradation of AMP to I M P takes place during a single muscle twitch. T h e
deamination and reamination steps seemed to be out of phase, deamination occurring
during contraction, whereas reamination is part of the recovery from exercise. Later
attempts to measure AMP-IMP conversion during a single muscle twitch radiochemically, however, were unsuccessful (Cain et af., I 963).
Operation of the P N C was finally demonstrated in a gel-filtered, dialysed high-speed
supernatant of rat muscle (Lowenstein & Tornheim, 1971; Lowenstein, 1972;
Tornheim & Lowenstein, 1972). T h e supernatant was incubated with IMP, GTP and
aspartate in the presence of a GTP-regenerating system (creatine phosphate and
creatine kinase). Under these conditions, I M P was aminated. A transient accumulation
of adenylosuccinate occurred, but eventually all I M P was converted to adenine
nucleotides. Ammonia production was negligible, due to inhibition of adenylate
deaminase by G T P . This condition mimicks resting muscle under aerobic conditions.
262
ARENVAN WAARDE
When 2-deoxyglucose and hexokinase were subsequently added to the extract,
adenine nucleotides were rapidly converted to IMP. A T P was used to phosphorylate
2-deoxyglucose in the hexokinase reaction. Since deoxyglucose cannot be used as a
substrate for glycolysis, high-energy phosphate became trapped in the form of
2-deoxyglucose 6-phosphate and accumulation of A M P occurred via the myokinase
reaction. AMP-deaminase was activated, due to an increased availability of substrate
and disappearance of G T P , whereas adenylosuccinate synthetase became rapidly
inhibited due to the formation of G D P . This situation simulates tetanic exercise.
By repeated addition of creatine phosphate and 2-deoxyglucose, complete amination
and deamination of the purine nucleotides was obtained 4 times, indicating the ability
of the P N C to operate as a cycle. Formation of ammonia was shown to be dependent
on the presence of aspartate and catalytic amounts of I M P (or ATP, or adenylosuccinate) in the muscle extract (Lowenstein, 1972; Tornheim & Lowenstein, 1972).
( 3 ) Proposed physiological functions of the cycle
In any discussion of the operation of the PNC, we should keep in mind that the cycle
consists of two components :
( a ) Deamination of AMP generates I M P and NH,. This part of the cycle has no
direct relationship with aerobic metabolism, but it may be particularly important
during glycolytic energy production.
(b) Reamination of I M P uses the amino group of aspartate and produces fumarate
and AMP. This pathway is closely linked to aerobic energy production.
Thus, the following functions of the P N C may be envisaged:
A d (4
( I ) Stabilization of the adenylate energy charge (ATP/ADP-ratio, or phosphorylation potential)” by the adenylate deaminase reaction in situations when energy
demand surpasses energy production.
This function has been proposed by Lowenstein (1972), Chapman & Atkinson (1973)
and Chapman et al. (1976). When liver cells (or the intact liver) are incubated with
fructose or 2-deoxyglucose, for example, inorganic phosphate becomes trapped in the
form of fructose- I -phosphate or 2-deoxyglucose-6-phosphate.Correct ATP/ADP
ratios can no longer be maintained by glycolysis and oxidative phosphorylation, since
the concentration of inorganic phosphate has dropped so much that it becomes rate-
* T h e energy charge of a cell is the extent to which the adenine nucleotide pool is ‘charged’ with high-energy
phosphate bonds. It is calculated by the formula
energy charge =
[ A TP] + O s [ADP]
[ATP]
+ [ADP] + [AMP]
’
T h e energy charge is equal to I when all adenine nucleotide is present in the form of A T P ; it is o when the pool
consists only of AMP. T h e charge concept was originally formulated by Atkinson (1968).An even more sensitive
indicator of the energy status of a cell is its phosphorylation potential :
phosphorylation potential =
[ATPI
[ADPI’ [Pi1
Both the energy charge and the phosphorylation potential are a measure for the ability of the cell to carry out
ATP-dependent processes.
The purine nucleotide cycle
263
limiting. In this case, adenylate deaminase is activated, resulting in a decrease of the size
of the adenylate pool. T h e ATP/ADP ratio is increased, since the myokinase
equilibrium (reaction 5) is pulled in the direction of ATP-formation :
zADP +AMP
AMP
+A T P
(5)
+ H,O + I M P + NH,
Net reaction : zADP + A T P
+ I M P +N H ,
(1)
(6)
(2) Control of the rate of glycolysis and glycogenolysis via allosteric modulation of
phosphofructokinase.
This function has been proposed by Lowenstein (1972) and Sugden & Newsholme
( I975). Ammonium ions stimulate phosphofructokinase (PFK), an enzyme that
catalyses one of the rate-limiting steps of glycolysis. T h e activation is not based on any
change in p H (Abrahams & Younathan, 1971). Lowenstein (1972) proposed that
ammonia liberated in the adenylate deaminase reaction might have a twofold effect:
(i) Direct activation of PFK.
(ii) Indirect activation of P F K by buffering of hydrogen ions (produced during
lactate formation). A rise in p H makes P F K much less susceptible to inhibition by its
allosteric effector ATP.
Ad ( b )
(3) Control of the total level of citric acid cycle intermediates (see Fig. I). Operation
of the P N C will increase the total level of T C A cycle intermediates because of the
conversion of aspartate to fumarate. Such an expansion of the pool of citric acid cycle
intermediates will lead to an activation of aerobic energy production, since four-carbon
' sparker' molecules are provided. This function was proposed by Lowenstein (1972)
and it may be particularly important in tissues which lack alternative anaplerotic
mechanisms.
(4) Liberation of ammonia from amino acids. When the net reaction of the cycle (4)
is coupled to the fumarase (7), malate dehydrogenase (8) and glutamate-oxaloacetate
transaminase (9) reactions, we obtain the following scheme :
+ G T P + H,O -+ fumarate + G D P + Pi + NH,
fumarate + H,O +malate
malate + NAD+ oxaloacetate + N A D H + H+
glutamate + oxaloacetate +alpha-ketoglutarate + aspartate
overall : glutamate + NAD' + G T P + 2H,O + alpha-ketoglutarate
+ N A D H + G D P + Pi + NH,'
aspartate
+
(4)
(7)
(8)
(9)
(10)
Reaction ( 1 0 ) is similar to the oxidation of glutamate by glutamate dehydrogenase ( I I ) ,
the only difference being the hydrolysis of the high-energy phosphate bond of
GTP:
glutamate
+ NAD'
+ alpha-ketoglutarate
+ N A D H + NH,'.
(10)
ARENVAN WAARDE
264
Cvtosol
Mitochondria
AcCoA
MAL-
t
-tf
OAA
MAL
CIT
\
I so
;partate
Fig. I . Anaplerotic role of the purine nucleotide cycle. Operation of the cycle results in the formation of
fumarate in the cytosolic compartment. Fumarate is converted to malate by fumarase and transported into
the mitochondria. The pool of four-carbon ‘sparker’ molecules (malate, oxaloacetate) is thus increased,
resulting in a more rapid oxidation of acetyl-coA by the Krebs cycle.
T h e free energy of reaction (10) is about - 1 . 0 kcal/mol, that of reaction ( X I )
approximately
6.5 kcal/mol (Lowenstein, 1972). The P N C is therefore poised
towards oxidative deamination of glutamate, whereas the glutamate dehydrogenase
reaction is poised towards reductive amination of alpha-ketoglutarate. Reaction ( I I ) can
only proceed in the indicated direction when the end products, alpha-ketoglutarate and
NADH, are rapidly catabolized.
As evidence to support this function, Lowenstein (1972) presented the following
data :
(i) Glutamate dehydrogenase and adenylate deaminase show reciprocal tissue
distributions, a high activity of the former being accompanied by low activity of the
latter and vice versa. This suggests glutamate dehydrogenase and the P N C to be
alternative pathways for amino acid deamination.
(ii) Mitochondria from a variety of tissues do not normally liberate ammonia from
glutamate. Glutamate catabolism proceeds in these cases via a transamination reaction
involving oxaloacetate with the resultant production of aspartate (see Borst, 1962; de
Haan et al., 1967; Krebs & Bellamy, 1960; Van Waarde & De Wilde-van Berge
Henegouwen, I 982). Ammonia production by these tissues therefore seems to require
extramitochondrial deamination of aspartate via the PNC.
(5) Control of the relative amounts of adenine and guanine nucleotides in the cell (see
Fig. 2 ) . Adenylates can be converted into guanylates by the action of AMP-deaminase,
I M P dehydrogenase and G M P synthetase. Guanylate/adenylate conversion involves
GMP-reductase, adenylosuccinate synthetase and adenylosuccinate lyase. G M P -
+
The purine nucleotide cycle
I
G DP
vt
/'
I
AMP
I
-
ATP
XMP
G
Pi
t
de novo
purine
synthesis
Fig. 2. Relationship between the purine nucleotide cycle and the interconversion of adenine and
guanine nucleotides. T h e numbered enzymes are: I , Adenylosuccinate synthetase (E.C. 6 . 3 . 4 . 4 ) ;
2, Adenylosuccinate lyase (E.C. 4 . 3 . 2 . 2 ) ;3 , AMP deaminase (E.C. 3 . 5 . 4 . 6 ) ;4. IMP dehydrogenase
(E.C. I . z . I . 14);5 , GMP synthetase (E.C. 6 . 3 . 5 . 2 ) ;6, GMP reductase (E.C. I . 6 . 6 . 8 ) .
synthetase requires ATP, whereas adenylosuccinate synthetase uses G T P . AMPdeaminase is inhibited by GTP and GMP-reductase activity is depressed by A T P
(Henderson & Paterson, 1973). These regulatory properties help the cell to maintain
the proper balance between the levels of adenine and guanine nucleotides.
11. LIVER
When D-fructose (10 mM) is infused in rats, striking metabolic changes occur in the
liver. Within 10 minutes fructose- I-phosphate accumulates to a level of 8.7 ,umol/g wet
weight. T h e ATP-content decreases to 23 yo of the control value, whereas I M P rises
sevenfold to a maximum of 1.1 ,umol/g (Woods et al., 1970).
Fructose- I -phosphate is formed by the activity of fructokinase (E.C. 2 . 7 . I .3) :
fructose
+ A T P + fructose- I -phosphate + ADP.
(12)
T h e high rate of this reaction causes a rapid decrease in the concentrations of A T P and
inorganic phosphate. Due to the decrease of the value of the adenylate energy charge
and the disappearance of inorganic phosphate, AMP-deaminase becomes activated and
AMP is deaminated to IMP. Fructose- I -phosphate is under normal conditions
removed by the action of aldolase (E.C. 4. I .2.13), which has an activity equal to that
of fructokinase :
fructose- I -phosphate + dihydroxyacetonephosphate
+ D-glyceraldehyde,
( I 3)
T h e accumulation of fructose- I -phosphate which occurs in the presence of exogenous
fructose therefore needs an explanation. According to Woods et al. (1970), aldolase is
inhibited by I M P and fructose- I -phosphate accumulation thus becomes a prolonged
phenomenon.
266
ARENVAN WAARDE
Chapman & Atkinson (1973) examined the kinetic properties of rat liver AMPdeaminase, which are responsible for the decline of the adenylate pool and the increase
of I M P in the presence of fructose. A partially purified preparation of the enzyme
proved to be activated by A T P under all conditions examined. T h e effect is largely on
the affinity of the enzyme for its substrate, K,,, values for A M P being lowered from I 1 . 1
to 0.3 mM in the presence of ATP. Liver AMP-deaminase is also activated b y ADP, but
to a lesser degree. T h e authors evaluated the response of the enzyme to variations in the
energy charge at a constant size of the adenylate pool. In the physiological range of
energy charge (o*-po*g),the rate of deamination increases sharply with decreasing
charge. When the charge drops below 0.6, however, the activity of the enzyme decreases
again. Chapman 8z Atkinson (1973) therefore suggested that liver AMP-deaminase
serves to protect the organ against transient decreases of the energy charge during
situations in which there is an imbalance between ATP-consumption and ATPproduction. T h e decrease of enzyme activity at levels of charge below 0.6 was explained
as a self-limiting response to prevent excessive depletion of the adenine nucleotide pool.
Protection of the cell against short-term decrease in energy charge is at the expense of
the adenine nucleotide pool, but the dependence of AMP-deaminase activity on the
adenylate pool size provides a built-in limit on how far the pool can be reduced. Because
of the limited amount of adenine nucleotides present in the cell, stabilization of the
energy charge by the adenylate deaminase reaction cannot be a long-term effect ; it can
protect only against sharp drops in charge during the transient period while a new
steady-state is established (in which ATP-formation and ATP-consumption are once
more equal).
Van den Berghe et al. (1977) made a detailed study of the metabolic changes
occurring in mouse liver after fructose loading. They showed that fructose- I -phosphate
accumulates during the first minute after the onset of fructose infusion, whereas IMPaccumulation occurs only during the second minute. Therefore, IMP-inhibition of
aldolase is not the mechanism responsible for fructose- I -phosphate accumulation. T h e
apparent blockade of aldolase seems to be caused by the fact that the following steps in
the glycolytic chain cannot keep pace with the fructokinase and aldolase reactions. On
the basis of metabolite measurements, Van den Berghe et al. ( I977) proposed that the
disappearance of G T P is one of the signals leading to deinhibition of AMP-deaminase
and IMP-accumulation. An additional mechanism might be the drop of the inorganic
phosphate concentration (Oberhansli et al., 1987).
When liver AMP-deaminase is inhibited by a high dose of coformycin ( 5 0 PM), the
fructose-induced breakdown of A T P is unaffected, but the depletion of the adenine
nucleotide pool proceeds much more slowly (Van den Berghe et al., 1980). There is a
strong increase in the level of AMP, whereas I M P does not accumulate at all.
S’-Nucleotidase seems to be inactive, unless the AMP-concentration rises to
unphysiological values. As a consequence, the drop of the liver adenylate energy charge
after fructose administration is much greater in coformycin-treated rats than in control
rats (Van den Berghe et al., 1980, see Table I).
A similar degradation of adenylates and accumulation of IMP due to activation of
AMP-deaminase is observed in liver cells during environmental anoxia (Vincent et al.,
1982).
T h e role of glutamate dehydrogenase and the P N C in urea formation from amino
The purine nucleotide cycle
Table
I.
267
Changes in metabolite levels (pmoleslg wet weight) of rat hepatocytes
after treatment wzth fructose
(Data of Van den Berghe et af. (1980).)
Control rats
Coformycin-treated rats
No fructose
Fructose z m
2.3
07
0Z+
0 1
0'4*
08
03*
0' 1
I.I*t
TAN
2'9
"5*
3' I
26
AEC
083
053*
0.85
029't
Parameter
No fructose
ATP
ADP
08
AMP
2'0
Fructose z m
1.3*t
TAN = Total level of adenine nucleotides; AEC = Adenylate energy charge.
* Significant difference between ' No fructose ' and fructose-treated groups.
t Significant difference between control and coformycin-treated rats.
acids has for some time been a subject of discussion. In mammals, urea is the end
product of protein catabolism and is synthesized from aspartate and ammonia by
carbamyl phosphate synthetase and the urea cycle. It has been generally believed that
the free ammonia which is necessary for urea formation arises from the action of
glutamate dehydrogenase in the mitochondria1 matrix (Lehninger, I 970).
This assumption was questioned during the mid-seventies in several publications.
Experiments using rat liver mitochondria suggested that the in vivo activity of
glutamate dehydrogenase is too low to account for the observed rates of urea synthesis,
due to a slow entry of glutamate in the mitochondrial matrix (see McGivan et al., 1974;
McGivan & Chappell, 1975). T h e amino acid leucine accelerates glutamate
dehydrogenase in sonicated mitochondria, but it inhibits urea synthesis in intact
hepatocytes. O n the basis of these observations, McGivan and co-workers suggested
that glutamate dehydrogenase does not play any role in liver amino acid deamination.
T h e enzyme would function primarily in the direction of glutamate formation as a
mechanism for nitrogen storage, whereas the ammonia necessary for urea synthesis
would originate in the cytoplasmic PNC (McGivan & Chappell, 1975; Moss &
McGivan, 1975; Mendes-Maurad et al., 1975; Sies et al., 1975).
However, the argumentation which was presented in support of a function of the
P N C in urea synthesis was subsequently invalidated by observations of Rognstad
(1977) and Krebs et al. (1978). Rognstad ( I977) demonstrated that leucine inhibition
of urea synthesis in intact hepatocytes can be overcome by administration of ornithine,
which suggests that the main site of leucine action is at an enzyme from the urea cycle,
ornithine transcarbamylase, rather than at glutamate dehydrogenase. He also showed
that the formation of glucose and lactate from asparagine in intact hepatocytes can be
strongly suppressed by addition of amino-oxyacetate, an inhibitor of transamination,
whereas hadacidin, an inhibitor of the purine nucleotide cycle, has no effect on urea
synthesis. Krebs et al. (1978) demonstrated that the initial rate of incorporation of
[I5N]alanine into the 6-amino group of adenine nucleotides in rat hepatocytes is about
one-eighteenth of the rate of incorporation into urea. These results suggest that,
268
ARENVAN WAARDE
contrary to the opinion of the previous authors, the P N C is not a major pathway for
ammonia production in rat liver. In the intact organ, glutamate dehydrogenase seems
to be perfectly able to sustain normal rates of amino acid deamination.
A similar conclusion is valid for the liver of ammoniotelic fish. In goldfish
hepatopancreas, activity of glutamate dehydrogenase (in the direction of oxidative
deamination) is r oo-fold greater than that of adenylosuccinate synthetase, the ratelimiting step of the P N C (Van Waarde, 1981). Endogenous ammonia production of
goldfish hepatocytes (corrected for net adenylate degradation) is unaffected by the
transaminase inhibitor amino-oxy-acetate or the PNC-inhibitor hadacidine (Van
Waarde & Kesbeke, 198 I 6). Ammonia production from endogenous leucine, however,
is completely blocked by amino-oxy-acetate and only slightly inhibited by hadacidine
(Van Waarde & Kesbeke, 1981b). These data suggest that amides (asparagine,
glutamine) and glutamate are the major endogenous ammoniogenic substrates (see also
French ~t al. 1981; Campbell et al., 1983), whereas glutamate dehydrogenase is the
enzyme involved in amino acid deamination (Van Waarde & Kesbeke, 19816).
Using "N-labelled amino acids, Casey et al. (1983) measured the rate of [I5N]incorporation into ammonia and the 6-amino group of the purine nucleotides by liver
cells of the channel catfish. T h e rate of appearance of the label in ammonia proved to
be much higher than in the purine amino group. Therefore, the P N C is not a significant
mechanism for amino acid deamination in fish hepatocytes. T h e evidence available for
livers of catfish, goldfish and other fish species indicates that the major route for amino
acid catabolism is the transdeamination pathway.
111. KIDNEY
A very important function of the kidney is the production of ammonia for
maintenance of the acid-base balance. In response to acid loads, excess protons are
combined with ammonia and excreted as ammonium ions. Production and excretion of
ammonia are therefore greatly elevated in metabolic acidosis.
It has been well established that most of the ammonia formed in kidney originates
from the amide and amino nitrogens of glutamine. T h e deamidation reaction which
converts glutamine to glutamate is known to be catalysed by phosphate-dependent
glutaminase (E.C. 3 . 5 . I .2),but the question, which route is followed for deamination
of glutamate, has been hotly debated in recent literature. Major candidates are the
glutamate dehydrogenase reaction (E.C. I . 4 . I . 2) and the PNC. T h e presence of the
latter pathway in rat kidney has been demonstrated b y Bogusky et al. (1976). During
ischaemia, I M P accumulates in kidney of mouse (Warnick & Lazarus, 1981) and rats
(Gerlach et al., 1963). Turnover of the 6-amino group of adenine nucleotides in kidney
is substantial, approximately 3-4 times greater than in liver.
T h e effect of acid feeding ( I 20 h) and recovery (48 h) on the metabolism of rat kidney
has been examined by Bogusky et al. (1981). Acidosis causes an 8-fold increase of the
level of IMP, whereas adenylosuccinate decreases to 25 7; of the control value. After
cessation of acid feeding, normal IMP-levels are rapidly restored, but adenylosuccinate
declines further and drops below the limit of detection. Kidney a-ketoglutarate content
decreases 9-fold during acidosis and returns to normal after termination of the acid
intake.
The activities of adenylosuccinate synthetase, adenylosuccinate lyase, glutaminase
The purine nucleotide cycle
249
and glutamate dehydrogenase are 2-2’5 fold increased during acidosis, whereas AMPdeaminase is unchanged. Activity of adenylosuccinate synthetase, the rate-limiting step
of the PNC, parallels the rise and fall in ammonia excretion, but glutaminase and
glutamate dehydrogenase remain elevated during at least 48 h of the recovery period,
when ammonia excretion has returned to the control rate.
The authors concluded that changes in the activity of the PNC correlate with changes
in ammonia excretion to a more parallel degree than do the activities of glutaminase or
glutamate dehydrogenase. They proposed that the PNC may play a regulatory role in
renal ammoniogenesis. It is evident, however, that the activity of adenylosuccinate
synthetase is quite minor compared to the high levels of glutaminase and glutamate
dehydrogenase observed under the same conditions.
A significant role of the PNC has also been proposed on the basis of experiments
using enzyme inhibitors (Bogusky et al., 1983).Isolated kidney tubules were incubated
with 2-amino-~-methoxy-trans-but-~-enoic
acid (MVG), an inhibitor of transamination. The drug has no effect on the activity of the enzymes of the PNC or on
glutamate dehydrogenase. In the presence of MVG, transamination is blocked so that
ammonia from exogenous aspartate can only be produced via the PNC. Ammonia
production from exogenous glutamate, however, can proceed only via the glutamate
dehydrogenase reaction. A comparison of the rates of ammonia production from
aspartate and glutamate under these conditions will therefore give information about
the relative capacity of the PNC and the glutamate dehydrogenase reaction to produce
ammonia in the intact kidney cell. T h e results showed that ammonia can be formed by
glutamate dehydrogenase and the PNC at significant and approximately equal rates. It
should be noted, however, that the experiments cannot be considered proof of
ammoniagenesis by the PNC in the normal situation. Although aspartate metabolism
must proceed via the PNC in the presence of MVG, in its absence the pathway may be
different. An inhibitor might merely divert the flow from a major to a normally minor
pathway.
Bogusky 8z Aoki (1983)studied ammonia production in the isolated perfused rat
kidney, using [15N]glutamate as a substrate. Before isolation of the kidney, rats were
treated with the glutamine synthetase inhibitor methionine sulfoximine to simplify
calculation of the nitrogen balance.
With 2 mM [15N]glutamate and 5 mM glucose as exogenous substrates, [15N]incorporation into the 6-amino group of adenine nucleotides seemed to be earlier and
to be much higher than the appearance of I5Ninto free ammonia. For this reason, the
authors assumed that glutamate deaminated by the PNC was the primary source of
ammonia.
When the tricarboxylic acid cycle was inhibited at the level of aconitase with 0.1mM
fluorocitrate, ammonia production was increased fourfold above the control level. From
simultaneous measurements of the levels of endogenous amino acids and purine
nucleotides, Bogusky 8z Aoki (I 983) concluded that also in this case, glutamate was the
main ammoniogenic substrate.
During the first 5 minutes after addition of fluorocitrate, both the turnover rate of the
6-amino group of adenine nucleotides and kidney IMP-content showed a more than
threefold increase, which was accompanied by a rapid fall in the tissue levels of
glutamate and aspartate. T h e authors therefore proposed that ammoniagenesis in the
10
RHE
63
270
ARENVAN WAARDE
presence of fluorocitrate occurs initially v i a the PNC. After 5 minutes, however, the
ATP-level showed a 50 Yo decline and IMP-content and the incorporation of 15N into
the purine amino group decreased rapidly. The PNC seemed to be inhibited and
ammonia formation proceeded now via the glutamate dehydrogenase reaction in
amounts stoichiometric with uptake of glutamate from the perfusate.
Ammonia could also be formed from exogenous aspartate at a rate of ~ . o p m o l /
min .g. After addition of fluorocitrate, ammonia production and renal uptake of
aspartate showed a parallel decline, but tissue levels and uptake rates of glutamine and
glutamate were unaffected. Since fluorocitrate inhibits the PNC, whereas glutamate
dehydrogenase is stimulated, Bogusky & Aoki (1983) concluded that all ammonia
produced from aspartate is formed via the PNC and that the PNC can make ammonia
from aspartate at a rate of 1.0pmollmin. g, which is equal to the rate of ammonia
formation by glutamate dehydrogenase.
It should be noted, however, that the conditions which were used in their study were
unphysiological, since methionine sulfoximine can be expected to have a variety of
effects on kidney nitrogen metabolism. It is also possible to challenge the evidence they
presented in support of a major function of the PNC in glutamate deamination. They
compared labelling of free ammonia and the purine amino group by examination of the
atom % excess 15N in both types of compounds. No correction was made for the pool
sizes of the metabolites. Since very high levels of unlabelled ammonia were present at
the beginning of the experiments and since the total amount of adenine nucleotides in
the kidney is relatively small, pool sizes have to be taken into consideration. When the
absolute amounts of “N present in ammonia and adenine nucleotides in Bogusky &
Aoki’s experiments are calculated from the atom % excess 15N and the respective pool
sizes, it can be shown that in reality, incorporation of label in ammonia was much higher
(and not lower) than in the purine amino group (see the discussion in Tornheim et al.,
1986). T h e conclusion that the P N C is the major pathway for kidney glutamate
deamination has therefore not been substantiated. Perfusion of a kidney with high levels
of aspartate creates an artificial situation, since in the intact animal plasma levels of
aspartate are very low.
Strzelecki et al. (1983) conducted studies with slices of rat kidney cortex, using
[‘6N]glutamine as substrate. ”N incorporation in the amino group of adenine
nucleotides proved to be negligible under these conditions. Addition of inorganic
phosphate (a strong inhibitor of AMP-deaminase) had no effect on ammonia formation
from glutamine. When intracellular Pi was lowered by administration of fructose (a
condition in which AMP-deaminase can be expected to be stimulated), ammonia
production rates were also unaltered. Strzelecki et al. ( I 983) therefore suggested that
the contribution of the P N C to ammonia formation from glutamine is insignificant. It
is possible, however, to deny the validity of that conclusion because of the unfavourable
energetic condition of the slices. Although no net adenylate degradation occurred
during the course of the experiment, the adenylate energy charge had the very low value
of 0.59 (Table 2 in their paper). Under such conditions, which differ vastly from the in
vivo situation, the PNC can be expected to be inhibited at the level of adenylosuccinate
synthetase.
Nissim et al. (1985, 1986) and Tornheim et al. (1986) studied the metabolism of
[“N]glutamine, [lSN]glutamate and [16N]aspartate in isolated kidney tubules. Tubules
The purine nucleotide cycle
271
are a much better preparation for the study of kidney metabolism than slices, since they
maintain levels of high-energy phosphates similar to those of the intact organ and are
capable of active ion transport. T h e intactness of the tubule preparation can be inferred
from the fact that the adenylate energy charge was maintained at a level of 0.83-0.87
during all experimental treatments (Nissim et al., 1986, calculated from their table 11).
Nissim et al. (1985) made a very detailed study of [“N]incorporation in many
metabolites (not only purine nucleotides and ammonia, but also alanine and aspartate),
using the sensitive technique of gas chromatography-mass spectrometry (GC-MS).
Tubules from chronically acidotic rats were compared to tubules isolated from normal
rats.
Experiments in which the amide group of glutamine was labelled showed that in
control animals approximately 90 76 of ammonia nitrogen arises by deamidation versus
60 yo in tubules from acidotic rats. Experiments in which the amino group of glutamine,
or the amino group of glutamate were labelled, indicated that in acidosis approximately
30 % of ammonia nitrogen is derived from the amino group by the activity of glutamate
dehydrogenase. Control rats did not form “NH, from [2-“N]glutamine. Transamination rates and transfer of “N to the amino group of purine nucleotides were
much higher in tubules from normal rats than in a preparation from chronically acidotic
rats. In tubules from acidotic rats, the total amount of 15N and the rate of appearance
of “N in free ammonia were much (i.e. 80-100 fold) higher than in the purine amino
group. Nissim et al. (1985) interpreted these data as showing that glutamate
dehydrogenase rather than the PNC is responsible for the increased ammoniagenesis
which occurs in acidosis. Since no precursor-product relationship was observed
between the 6-amino group of purine nucleotides and free ammonia, the contribution
of the PNC to ammoniagenesis appears to be relatively unimportant in chronically
acidotic rats. In tubules from normal rats incubated with [2-”N]glutamine, however,
the isotopic enrichment of the 6-amino group of purine nucleotides was higher and
faster than that of free ammonia. In normal acid-base status, the turnover of the adenine
nucleotides seems therefore to be able to account for the ammonia formed from
glutamate (Nissim et al., 1985).
In order to evaluate further the operation of the PNC, Nissim et al. (1986) incubated
tubules with [“N]aspartate and 5-amino-4-imidazolecarboxamideriboside (AICAriboside), a precursor of IMP. In tubules isolated from control rats, low levels of
AICAriboside ( I mM) significantly stimulated production of labelled ammonia and
incorporation of label in the amino group of purine nucleotides. T h e stimulation of
ammonia production from aspartate probably took place via the P N C rather than the
glutamate dehydrogenase reaction, since the rate of [“N]glutamate formation from
[“Nlaspartate was not changed by AICAriboside. However, a high dose (5 mM) of the
drug inhibited ammonia production and reduced tubule purine nucleotide content.
This biphasic influence of the riboside can be explained on the basis of its welldocumented effects on purine metabolism (see, e.g. Sabina et al., 1982; Swain et al.,
1984). Low doses of AICAriboside stimulate the PNC by increasing the availability of
IMP. At high doses, however, intracellular AICAribotide accumulates and
adenylosuccinate lyase, one of the enzymes of the PNC, is inhibited. In tubules from
acidotic rats, isotopic enrichment in ammonia was higher and faster than in the 6-amino
group of the adenine nucleotides, i.e. no precursor-product relationship was observed
272
ARENVAN WAARDE
between the nucleotides and ammonia. In tubules from control rats, however,
enrichment in the 6-amino group of adenine nucleotides exceeded that in ammonia.
T h e mentioned relationships were observed whether or not AICAriboside was included
in the incubation medium. T h e experiments of Nissim et al. (1986) therefore suggest
that during normal acid-base balance, the P N C plays a small role in ammoniagenesis.
Even in the acidotic state some ammonia seems to be formed by the PNC.
Somewhat different conclusions were reached by Tornheim et al. (1986), who
incubated renal cortical tubules (isolated from acidotic rats) with [2-''N]glutamine,
[16N]glutamate and [''Nlaspartate. They found no ( < IoO/") incorporation of label into
adenine nucleotides, even after prolonged incubation ( I h), although the energy charge
of the tubules was normal. From the measured concentrations of adenine nucleotides
and ammonia, and the labelling of ammonia, Tornheim et al. ( I 986) calculated that the
of the deamination of alpha-amino
flux through the P N C accounted for less than I
groups from all three substrates. Thus, the P N C was considered insignificant for
ammoniagenesis in tubules from acidotic rats. In tubules from normal rats there was a
greater incorporation of label into adenine nucleotides and amino acids, accompanied
by a reduced release of ammonia. Therefore, the P N C seems to play some role in basal
ammoniagenesis.
In summary, it can be said that the PNC is not the major deaminating pathway in
vertebrate kidney. Some cycling does occur, however, especially in the situation of
normal acid-base balance.
IV. PLATELETS
AMP-deaminase activity of human platelets can be almost totally blocked by a 6 h
incubation in the presence of 200 ,UM coformycin (Ashby et al. 1983; Ashby & Holmsen,
1983). Addition of thrombin to a platelet suspension induces shape change, secretion
and aggregation of the cells. Secretion is accompanied by purine breakdown via AMPdeaminase and a burst of glycolysis ending in lactate production. Blocking of the
deaminase has no effect on thrombin-induced glycolysis or glycogenolysis (Table 2).
Even when the glycolytic pathway is maximally activated because of treatment of the
cells with antimycin A (an inhibitor of oxidative phosphorylation), deaminase blocking
has no effect on glycolytic rate (Table 2). These observations argue against involvement
of AMP-deaminase in a regulatory mechanism of glycolysis (either by ammonia
activation of phosphofructokinase or AMP-activation of phosphorylase).
When untreated platelets are incubated with thrombin to induce secretion, the
adenylate energy charge remains stable at a level of 0.91f0.02,although up to 30 Yo of
the adenine nucleotide is converted to hypoxanthine. In coformycin-treated platelets,
an initial drop of the energy charge from 0.91 to 0.85 3 0 s after stimulation is followed
by a return to the control level during the next minute, Since almost no adenylate
catabolism takes place in these cells, the energy charge seems to be stabilized by ADPactivation of mitochondria1 ATP-synthesis (Ashby et al., 1983).
If hydrogen peroxide is added to a platelet suspension, a strong energy drain occurs,
resulting in an irreversible depletion of A T P and accumulation of hypoxanthine.
Hydrogen peroxide probably oxidizes intracellular glutathione, which has to be
resynthesized at the expense of NADPH. T h e decrease of NADPH apparently leads to
a powerful stimulation of the hexose monophosphate shunt. Under these conditions,
The purine nucleotide cycle
Table
2.
273
Lactate production and glycogenolysis in platelets
Untreated
AMP-deaminase blocked
-
Condition
Lactate
production
( 5 mM extracellular
glucose added)
Control
Respiration blocked by
antimycin A
Thrombin-stimulated
Thrombin antimycin A
1'7
+
Data presented as ,umol/min.
from Ashby et al. (1983).
toll
Glycogen
degradation
(no extracellular
glucose added)
< 1'0
~~
..
Lactate
production
(5 mM extracellular
glucose added)
1'5
I 2.5
Glycogen
degradation
(no extracellular
glucose added)
< 1'0
I 2.5
n.d.
6.0
'3'7
70'0
6.0
70'0
n.d.
13'4
n.d.
n.d.
cells (glycogen degradation as glucose units). n.d. = not determined. Data
the adenylate energy charge of untreated cells drops from a level of 0.90 to 0.83 after
1 0 min. However, in coformycin-treated cells a much larger drop from 0.89 to 0.71 is
observed over the same period. Whereas coformycin treatment in the absence of
H,O, has no influence on thrombin-induced platelet secretion, a drop of the adenylate
energy charge below 0.84 causes significant inhibition of secretion (Ashby et al., 1983).
Therefore, platelets treated with H,O, and coformycin show more inhibition of
thrombin-induced secretion than platelets treated with H,O, only. T h e role of platelet
AMP-deaminase seems to be stabilization of the adenylate energy charge in the absence
of stimulated oxidative phosphorylation or buffering of the energy charge during a
temporary imbalance between energy demand and energy production, until
mitochondria1 ATP-synthesis is sufficiently activated.
V. BRAIN
Neurons produce ammonia under a variety of conditions, such as electrical activity
and ischaemia. Ammoniagenesis is raised after the administration of convulsive agents
(Folbergrova et al., 1969; Richter & Dawson, 1949),whereas ammonia disappears from
the brain during sleep or anaesthesia. Since ammonia does not arise from activity of
glutaminase (Weil-Malherbe & Green, 1955 ; Folbergrova et al., 1969), the glutamate
dehydrogenase reaction and the P N C are the major candidates for deamination in the
central nervous system.
Schultz 8z Lowenstein (1976) demonstrated the occurrence of the reactions of the
PNC in a gel-filtered, high-speed supernatant of a rat brain homogenate. T h e reaction
was started by addition of I M P , G T P , aspartate and a GTP-regenerating system
(phosphoenolpyruvate with endogenous pyruvate kinase) to the extract. Nucleotide
interconversions were monitored by repeated spectral scanning in the range of
240-3 1 0nm and by enzymic analysis. Under these conditions, up to 80 yo of the I M P
was converted into ATP. After 75 min the supply of phosphoenolpyruvate became
exhausted, leading to a buildup of G D P and inhibition of adenylosuccinate synthetase.
A T P was then dephosphorylated by endogenous phosphatases and the resulting A M P
was deaminated by adenylate deaminase. Thus, the first turn of the PNC was
completed.
ARENVAN WAARDE
274
A problem in experiments with brain homogenates (as compared to muscle extracts)
is the presence of fairly high levels of S’-nucleotidase (14), adenosine deaminase ( I 5),
nucleoside phosphorylase ( I6) and guanase (17). T h e following reactions therefore
occur in the extract:
+ H,O + adenosine (inosine, guanosine) + Pi,
adenosine + H,O + inosine + NH,,
inosine (guanosine) +Pi +hypoxanthine (guanine) + ribose- I -P,
guanine + H,O + xanthine + NH,.
AMP (IMP, GMP)
(14)
(15)
(16)
(17)
As long as an excess of the GTP(ATP)-regenerating system is present, hypoxanthine
and guanine can be converted back to I M P and GMP, due to the activity of the enzymes
hypoxanthine (guanine) phosphoribosyltransferase ( I 8), phosphoribosylpyrophosphate
synthetase (19) and phosphoribomutase (20) :
+ PPriboseP + I M P (GMP) + PP,,
A T P + ribose- I-P + PPriboseP + AMP,
hypoxanthine (guanine)
ribose- I -P + ribose-5-P.
(18)
(19)
(20)
Xanthine, however, cannot be converted back to GMP. This makes demonstration of
repeated cycling of purine nucleotides more difficult in brain homogenates than it is in
muscle homogenates. T h e presence of adenosine deaminase ( I 5) and guanase ( I 7) in the
extract complicates calculations of ammonia stoichiometry, since these reactions form
ammonia as well as adenylate deaminase.
T h e maximum rate of ammonia formation observed in brain after administration of
convulsive agents (7.5 pmol/min .g, Folbergrova et al., 1969; Richter & Dawson, 1948)
is within the capacity of adenylate deaminase (8.3 pmollmin .g, Schultz & Lowenstein,
1976), but above that of glutamate dehydrogenase (3.5 pmol/min .g in the direction of
deamination at its pH-optimum of 8.0 with NAD as cofactor, Schultz & Lowenstein,
I 976). Steady-state rates of brain ammoniagenesis (0.037 pmol/min . g in rat brain,
0.13 pmol/min . g in guinea-pig brain, Weil-Malherbe & Green, 1955) compare
favourably with the maximum activity of adenylosuccinate synthetase (0.058 pmol/
min.g in rat brain, Schultz & Lowenstein, 1976).
Schultz & Lowenstein ( I976) therefore proposed that the P N C plays a significant role
in ammonia production by nerve cells. It may account for at least one-half of the
ammonia production in brain slices in the absence of exogenous substrates.
In a later series of experiments, Schultz & Lowenstein (1978) induced simultaneous
firing of as many neurons as possible by electrical shock treatment of rat brain in situ.
Subsequently, the brain was removed by freeze-blowing and metabolites were extracted
and assayed using standard biochemical techniques.
During 1 0s of shock treatment, a large drop in creatine phosphate (to < 20 Yo of the
control) and A T P (to 60 Yo of the control) was accompanied by an increase of ammonia.
Ammonia levels continued to rise until after I min a plateau of 3 times the control value
was reached. T h e decrease of A T P was accompanied by an increase of ADP, AMP and
IMP. Rephosphorylation began immediately after the electrical stimulus was turned off
The purine nucleotide cycle
27 5
as shown by the increase of creatine phosphate. Ammonia formation coincided with the
increase in AMP content and the appearance of IMP and adenylosuccinate. As soon as
AMP started to decline, ammoniagenesis came to a stop. Ammonia and IMP did not
accumulate in stoichiometric amounts, however, ammonia production being much
higher than the accumulation of IMP. The discrepancy between the observed IMP and
ammonia levels can be explained by removal of IMP via the adenylosuccinate
synthetase and 5’-nucleotidase reactions. It is possible, however, that in reality the
discrepancy was higher than observed, since part of the ammonia formed may have
been removed by the action of glutamate dehydrogenase and glutamine synthetase.
During the period of ammonia formation there was no decline of brain glutamate or any
increase of the mitochondria1 NADH/NAD+-ratio, whereas a z-s-fold decline of
a-ketoglutarate occurred.
In summary, Schultz & Lowenstein’s (1978) data show that a large portion of the
ammonia produced in the brain during shock treatment is due to the operation of the
PNC. A small contribution by the glutamate dehydrogenase reaction cannot be ruled
out, however.
VI. VERTEBRATE SKELETAL M U S C L E
Numerous observations suggest that the PNC serves an important function in
skeletal muscle :
(a) A unique isozyme of AMP-deaminase is found only in skeletal muscle (Ogasawara
et al., 1975, 1978; Solano & Coffee, 1978).
(b) AMP-deaminase activity is an order of magnitude greater in skeletal muscle than
in other organs, like brain, liver or kidney. This has been observed in many organisms,
including rat (Purzycka, 1962; Lowenstein, 1972; Chandrasena & Hird, 1978), rabbit
(Conway & Cooke, 1939), goldfish (Van Waarde, 1981a) and trout (Walton & Cowey,
1977).
( c ) Vigorous contraction of fast-twitch glycolytic fibres (but not slow-twitch fibres !)
is associated with the accumulation of stoichiometric amounts of IMP and NH, (Gerez
& Kirsten, 1965; Driedzic & Hochachka, 1976; Goodman & Lowenstein, 1977; Meyer
& Terjung, 1979, 1980; Meyer et al., 1980; Van Waarde & Kesbeke, 1983). Ammonia
subsequently appears in the efferent venous blood (Wilkerson et al., 1977; Babij et al.,
1983; Mutch & Banister, 1983). The activity of muscle AMP-deaminase is under tight
control, which appears to be both of a covalent and a non-covalent nature. Nucleotides
(ATP, ADP and GTP), inorganic phosphate and inorganic pyrophosphate are effectors
(Coffee & Solano, 1977; Ronca-Testoni et al., 1970; Wheeler & Lowenstein, 1979a, 6 ;
Van Waarde & Kesbeke, 1981a), whereas a covalent activation mechanism has been
described by Rahim et al. ( I 979).
(d) The muscle isozyme of AMP-deaminase binds to myosin. In rabbit muscle, the
binding has been reported to take place mainly at the heavy meromyosin fragment
(Ashby & Frieden, 1977, 1978; Barshop & Frieden, 1984; Koretz & Frieden, 1980;
Roretz, 1982), but in rat muscle it seems to occur at the light meromyosin fragment
(Shiraki et al., 1 9 7 9 ~ )The
.
association is very tight (Kd 60 nM, Shiraki et al., 1 9 7 9 ~ ) .
Binding leads to a substantial increase of deaminase activity, but actomyosin ATPase
is unaffected (Shiraki et al., 1979b). After 3 0 s stimulation of rat muscle, the ratio of
bound to free enzyme is raised fivefold above that in resting muscle. Stimulation also
276
ARENVAN WAARDE
increases the ammonia content of the muscle to 5 times that in resting state. Thus, a
correlation seems to exist between AMP-deaminase binding and muscular ammonia
production (Shiraki et al., I 98 I ) . Histochemical staining substantiates the association of
enzyme activity with the myofibrils (Ashby et al., 1979).
( e ) One form of adenylosuccinate synthetase is found predominantly in muscle
(Ogawa et al., 1977; Matsuda et al., 1977).
(f)Adenylosuccinate synthetase binds to muscle actin (Ogawa et al., 1978).
(g) Expansion of the pool of citric acid cycle intermediates occurs during muscle
contraction, due to aspartate deamination via the adenylosuccinate synthetase and
adenylosuccinase reactions (Aragon et al., 1980, 1981).Fumarate thus formed serves as
a 'sparker' in the tricarboxylic acid cycle (Scislowski et al., 1982).
( I t ) Adaptive increases in the level of muscle AMP-deaminase during training have
been reported (Hryniewiecki, 1971),although these results could not be confirmed in
later investigations (Winder et al., 1974).
(i) Changes occur in the muscle AMP-deaminase isozyme pattern during development. T h e appearance of the adult isozyme correlates with the onset of movement
(Kendrick-Jones & Perry, 1967; Sammons & Chilson, 1978).
More studies have been performed on the operation of the P N C in skeletal muscle than
in any other tissue.
One of the ways to assess the function of a metabolic pathway is by the study of
mutants, in which a gene coding for an enzyme from the pathway has been deleted.
Such a genetic defect does exist in the case of the PNC. A disease of human muscle,
myoadenylate deaminase deficiency (MDD) is caused by deletion of the gene that codes
for the muscle isozyme of adenylate deaminase (Fishbein et al., 1978, 1979). As a
consequence, patients have very low levels of AMP-deaminase in their muscles,
whereas other tissues (e.g. erythrocytes, leukocytes, fibroblasts) show normal enzyme
activity (Engel et al., 1964; Fishbein et al., 1978, 1980a, b ; Shumate et al., 1979, 1980;
DiMauro et al., 1980; Heffner, 1980; Scholte et al., 1981; Mercelis et al., 1981 ; Kar &
Pearson, 1981; Hayes et al., 1982; Kelemen et al., 1982). Study of these patients can
give interesting clues to the function of the P N C in the contraction process. Generally
observed symptoms are weakness (Fishbean et al., 1978; Shumate et al., 1979, 1980;
Kar & Pearson, 1981; Mercelis et al., 1981) besides cramping (Fishbean et al., 1978;
DiMauro et al., 1980; Kar & Pearson, 1981),stiffness (Hayes et al., 1982) and muscle
aches (Shumate et al., 1979; DiMauro et al., 1980; Kar & Pearson, 1981 ; Hayes et al.,
1982; Kelemen et al., 1982) after exercise. Although the existence of a causal
relationship between the enzyme defect and the clinical symptoms was initially denied
(Shumate et al., 1979, 1980), a relation between the gene deletion and post-exercise
myalgia seems to be established (Kelemen et al., 1982). The exact genetic nature of the
disease is still unclear, but the genets) involved seem to be localized in the autosomes
(Kelemen et al., 1982).
In a detailed study of the metabolic consequences of this deficiency, Sabina et al.
(1984) made patients and normal subjects perform exercise until exhaustion. They
noted the following symptoms :
ti) M D D patients suffer from easy fatigability. They can do only 28"/0 of the
The purine nucleotide cycle
277
maximal work of their non-MDD counterparts. Both the total work performed and
endurance are significantly lower in people suffering from M D D .
(ii) Although the decrease of the level of creatine phosphate during exercise is similar
in both groups, the median decrease in total phosphagen per unit work is thus fivefold
greater in M D D patients than in the non-MDD group.
(iii) M D D patients suffer from postexertional muscle aches, cramps and pains.
(iv) Lactate production during ischaemia is normal, but there is hardly any I M P
accumulation and a strongly depressed ammoniagenesis during ischaemic exercise.
(v) Exercising M D D patients produce much more adenosine than their non-MDD
counterparts. In vitro studies of nucleotide catabolism using the adenosine deaminase
inhibitor 2’-deoxycoformycin (DCF) suggest that the normal pathway for adenylate
breakdown (AMP + I M P -+ Ino + Hx) is replaced by an alternative route (AMP -+
Ado + Ino + Hx) in M D D patients. As a consequence, Hx-formation is inhibited by
D C F in the patient muscles, whereas in normal subjects, adenylate breakdown is
unaffected by DCF.
(vi) In the non-MDD group, a significant decrease of A T P occurs due to conversion
of adenylates to IMP. However, in M D D patients there is hardly any decrease of A T P
during muscle work.
In theory, the following symptoms could be expected to result from absence of
myoadenylate deaminase :
( I ) T h e decrease of ATP-content in skeletal muscle during exercise is normally not
associated with an increase in ADP or AMP because AMP-deaminase is activated when
the energy charge of the cell falls. Deamination not only prevents buildup of AMP, but
also the production of ADP, since the equilibrium of the myokinase reaction is pulled
towards A T P formation (see the Introduction). T h e net effect of the coupled reactions
is maximal extraction of energy from the adenylate pool. T h e adenylate energy charge
and A T P phosphorylation potential are kept at the maximum level during a temporary
imbalance between energy demand and energy production.
When AMP-deaminase is lacking, IMP-formation cannot occur, A D P and AMP can
therefore be expected to increase. Failure to extract all of the energy stored in the
adenylate pool might lead to inhibition of actomyosin ATPase and contribute to the
more rapid onset of fatigue in the patients (see Sahlin et al., 1978).
This explanation for MDD’s symptoms, however, is refuted by the data presented in
Table 3. T h e fall of the energy charge during exhaustive exercise is similar in normal
subjects and MDD patients. Neither normal humans nor the patient group show a
significant increase of ADP or AMP during muscular work. It is of course possible that
due to compartmentation, total pool data do not give adequate information about the
actual energy charge and ADP or AMP concentrations close to the actomyosin cross
bridges. On the basis of these data, however, it seems unlikely that major differences
would exist.
(2) AMP is normally deaminated to I M P during a heavy work load. Since I M P is a
charged compound, it cannot pass the plasma membrane or leak out to the circulation.
During recovery, I M P is rapidly reaminated and incorporated in the adenine nucleotide
pool. In muscles lacking AMP-deaminase, AMP is probably dephosphorylated during
exercise. T h e resulting adenosine leaks out to the circulation, leading to a loss of
ARENVAN WAARDE
278
Table 3. Changes in the muscle adenylate pool of normal subjects and patients suflering from
myoadenylate deaminase deficiency (MDD) during exhaustive exercise (data calculated
from Sabina et al., 1984)
Parameter
Normal subjects
MDD patients
Control
IMP
148.7f 1 2 s (6)
20.9 f 5'5 (8)
0 4 6 f 0 1 3 (6)
0 . 3 z f o . 1 9 (6)
EC
0.93 f o o z (8)
ATP
ADP
AMP
126.8k 16.7 (4)
20.9
*
2.4 (4)
089 (2)
0 0 6 f o ~ o r(3)
0 9 3 f0'01 (4)
Exercise
ATP
107'6f 1 5 . 0 (6)
ADP
z 1 . 9 f s ' z (8)
AMP
IMP
0 s s k 0 . 1 7 (6)
5 2 ' 4 f 1 4 2 (7)
EC
0 9 0 f0.02 (8)
117'7fr8.1 (4)
24'6 f 2.3 (4)
1.09 (2)
4 2 * 1'5
(4)
090f0.01 (4)
Values expressed as means f standard deviation (nmoles/pmole total creatine). Values for EC, the adenylate
energy charge, are without dimension. The only significant difference ( P < 0 0 5 ) is that between the IMP-contents
of normal muscles and patient muscles after exercise.
precursors for purine salvage. It might therefore be envisaged that in M D D patients,
restoration of the adenylate pool after exercise will be slower than in normal subjects.
This explanation for MDD's symptoms is also refuted by Table 3. Both in normal
subjects and M D D patients there is no significant decline of the muscle nucleotide pool
(ATP ADP AMP IMP) during exercise.
(3) Ammonia has been suggested to play a role in the activation of glycolysis due to
its stimulating effect on phosphofructokinase (Abrahams & Younathan, 197I ;
Lowenstein, 1972; Otto et al., 1976; Sugden & Newsholme, 1975; Tejwani et al.,
I 973). A significant correlation between AMP-deaminase activity and phosphofructokinase activity exists in different muscle types (Winder et al., 1974). It might
therefore be expected that lack of ammonia will lead to a delayed or impaired activation
of glycolysis in muscles of M D D patients. This explanation of the clinical symptoms
seems to be ruled out by the fact that lactate production during exercise is identical in
normal subjects and patients (DiMauro et al., 1980; Mercelis et al., 1981 ; Sabina et al.,
1984), although in one individual, a subnormal rise of blood lactate was observed
(Hayes et al., 1982). It is possible, however, that a defect of carbohydrate metabolism
other than M D D was responsible for impaired glycolysis in this patient (Hayes et al.,
I 982).
(4) Since AMP is an allosteric activator of phosphorylase b and phosphofructokinase,
whereas I M P is much less effective, accumulation of AMP in M D D patients during
exercise might lead to excessive glycogenolysis and a delayed 'switching off' of the
glycolytic pathway after termination of contraction (see Davuluri et al., 1981).This
explanation for MDD's symptoms seems also unlikely, since supranormal lactate
formation in M D D patients does not occur (DiMauro et d . ,1980;Mercelis et al., 1981 ;
Sabina et al., 1984).T h e increase in AMP concentration during the work load is minor
+
+
+
The purine nucleotide cycle
279
(see Table 3) and AMP is probably very rapidly phosphorylated after cessation of
exercise.
(5) Ammonia produced in the AMP-deaminase reaction could serve as a buffer for
H+ arising from ATP-hydrolysis (Hochachka & Mommsen, I 983) or lactate formation
(Lowenstein, I 972). Loss of buffering capacity in myoadenylate deficiency might cause
inhibition of actomyosin ATP-ase. This explanation of M D D symptoms is unlikely in
view of the vast amount of protons produced during exercise which is accompanied by
only minor ammoniagenesis. Lactate production in humans exceeds ammonia
production 30- to roo-fold (Wilkerson et al., 1977;Fishbein et al., 1978;Babij et al.,
1983;Katz et al., 1986).T h e contribution of ammonia to pH-buffering in muscle seems
therefore to be insignificant.
(6) Because lactate production is similar in normal and M D D subjects, whereas the
decrease in creatine phosphate per unit work is fivefold greater in the patients, it seems
that patient muscles have a lower capacity for aerobic energy production. Evidence that
blocking of the P N C gives rise to an impairment of aerobic energy generation also
comes from an animal model in which rats were infused with the drug 5-amino-4imidazolecarboxamide riboside (AICAriboside ; Swain et al., 1984;Flanagan et al.,
I 986).
AICAriboside is not a substrate for nucleoside phosphorylase and therefore does not
undergo any detectable phosphorolysis. T h e compound is phosphorylated to the purine
de n o w intermediate 5-amino-4-imidazolecarboxamideribotide (AICAR) by adenosine
kinase, AICAR is subsequently formylated to yield I M P (Lowy & Williams, 1977;
Zimmerman et al., 1978;Sabina et al., 1982).
When infused in rather low doses, AICAriboside increases the availability of IMP.
It can therefore be successfully used to enhance the rate of repletion of the A T P and
G T P pools in postischaemic myocardium (Swain e t al., 1982). At higher doses,
however, AICAR accumulates which is a strong inhibitor of adenylosuccinate lyase
(Sabina et al., 1982). Besides AICAR, 5-amino-4-imidazolecarboxamide ribotide
triphosphate (ZTP) is formed, since the pyrophosphate group of 5-phosphoribosyl- I pyrophosphate is transferred to AICAR in a reaction catalysed by 5-phosphoribosyl- I pyrophosphate synthetase (Zimmerman et al., 1978;Sabina e t al., 1983,1984,1985).
As a consequence, the aminating steps of the P N C are inhibited.
During both moderate (aerobic) exercise and severe (anaerobic) exercise, animals
treated with a high dose of AICAriboside generate the same initial force as control rats.
However, a decrease in tension occurs much more rapidly in AICAriboside-treated
rats. The total tension developed during the 10min experimental period is thus much
lower in AICAriboside-treated animals than in untreated controls. Anaerobic energy
production is unaffected, since lactate formation is identical in both groups.
AICAriboside-treatment therefore seems to depress aerobic energy production, which
occurs continuously during moderate exercise and initially during severe exercise.
On the basis of metabolite measurements in untreated and AICAriboside-treated
animals, Swain et al. (1984)and Flanagan et al. (1986)concluded that AICAriboside
prevents the increase in the activities of adenylosuccinate synthetase and adenylosuccinate lyase which normally takes place during exercise. T h e effects of such a
blockade (rapid fatigability, less total tension developed, more phosphagen breakdown
per unit workload, normal lactate production) show a striking resemblance to the
280
ARENVAN WAARDE
clinical symptoms of myoadenylate deaminase deficiency (MDD). The major effect of
a disruption of the P N C appears to be impairment of aerobic energy production due to
lack of four-carbon intermediates in the T C A cycle.
Essential to this explanation is the concept that the three enzymes of the P N C can be
simultaneously active in exercising muscle. Concurrent flux through AMP-deaminase
and the adenylosuccinate pathway was implied in the studies of Goodman & Lowenstein
(1977) and Lowenstein & Goodman (1978), who noted that exercise results not only in
an increase of the level of IMP, but also of that of adenylosuccinate. Simultaneous
action of the deaminating and reaminating parts of the cycle was denied by Meyer &
Terjung ( I 980), however. Based on experiments with the adenylosuccinate synthetase
inhibitor hadacidin, Meyer & Terjung (1980) and Meyer el al. (1980) concluded that
in working red muscle hardly any IMP-reamination occurs, whereas in white muscle
reamination is limited to the initial 5 min of a 3 0 min period of moderate exercise.
Meyer & Terjung ( I 980) therefore suggested that deamination takes place during
exercise and reamination occurs almost exclusively during recovery. Although Aragon
& Lowenstein ( I 980) used an identical experimental approach, they reached a different
conclusion. Aragon & Lowenstein (1980)followed changes in the levels of tricarboxylic
acid cycle intermediates in skeletal muscle of normal and hadacidin-treated rats. T h e
total level of metabolites from the citric acid cycle was found to rise by a factor of two
during exercise. Fumarate and malate reached their maximum first, followed by citrate,
isocitrate and succinate. These results suggest that fumarate or malate is the endproduct
of the anaplerotic reaction occurring under these conditions. Hadacidin had no effect
on the levels of Krebs cycle-intermediates in resting muscle, but it caused significant
inhibition of the rise in T C A cycle intermediates which occurs during exercise. From
simultaneous measurements of nucleotide levels, Aragon & Lowenstein ( I980)
concluded that the P N C is responsible for at least 72y0 (and probably more) of the
expansion of the citric acid cycle pool during exercise. In contrast to the opinion of
Meyer & Terjung (1980), Aragon & Lowenstein (1980) therefore stated that the P N C
is a major anaplerotic pathway in exercising rat muscle.
In a very interesting series of experiments, Manfredi & Holmes ( I 984) incubated gelfiltered protein extracts of rat skeletal muscle with a purine nucleotide pool of
physiological composition and size. An ATP-regenerating system or a substrate for
energy production were not included. In this in vitro model, the activities of the
individual PNC-enzymes and the effect of the composition of the nucleotide pool on
these activities could be evaluated. Surprisingly, the activity of each cycle enzyme was
observed to increase as the energy charge of the system was lowered over the range
0.99-0.64 (corresponding to a change of the ATP/ADP-ratio of 50 to I ) . T h e activities
of the PNC-enzymes increased in parallel with lowering of the charge and the
components of the cycle appeared to function as a coordinated unit. Similar results were
obtained whether an extract of fast (gastrocnemius) or slow (soleus) muscle was used.
The activation of AMP-deaminase at decreasing charge was to be expected, since GTP
is an inhibitor whereas ADP is an activator and A M P is the substrate (Ronca-Testoni
et al., 1970; Coffee & Solano, 1977; Wheeler & Lowenstein, 1979; Van Waarde &
Kesbeke, 198I a ) , but the activation of adenylosuccinate synthetase was unexpected,
since the purified enzyme is inhibited by G D P and ADP (Clark et al., 1977; Faraldo
et al., 1 9 8 3 ; Matsuda et al., 1977; Muirhead & Bishop, 1974; Ogawa et al., 1977; Van
The purine nucleotide cycle
28 I
der Weyden & Kelly, 1974). In the presence of a physiological purine nucleotide pool,
however, the availability of I M P seems to be the primary factor determining
activity.
Manfredi’s and Holmes’ experiments demonstrated not only that the aminating and
deaminating parts of the cycle can be simultaneously active, but they provided also
strong evidence for stabilization of the energy charge by the PNC. Regardless at which
value of the energy charge the incubation was started (0.99-0.69), the charge was
eventually stabilized at a level of 0.94-0.97. By use of the AMP-deaminase inhibitor
coformycin, the authors showed that AMP-deaminase pulled the myokinase equilibrium towards ATP-formation, this mechanism being responsible for stabilization of
the energy charge (Manfredi & Holmes, 1984). A function of the P N C in the
maintenance of a high ATP/ADP ratio has also been implied by the in vitro studies of
Tornheim & Lowenstein (1973, 1974, 1975 ; reviewed by Tornheim, 1979).
T h e P N C is not the only reaction which can supply carboxylic acids to the Krebs
cycle during exercise. In theory, the following reactions may also be involved.
(i) Alanine aminotransferase (E.C. 2 . 6 . I .2 , reaction Z I ) , or the concerted action of
alanine aminotransferase and aspartate aminotransferase (E.C. 2 . 6 . I . I , reaction 9) :
+
+
pyruvate glutamate +alpha-ketoglutarate alanine
+
+
aspartate alpha-ketoglutarate +glutamate oxaloacetate
Net : pyruvate
+ aspartate +alanine + oxaloacetate
(21)
(9)
(22)
Reaction (2I ) produces alpha-ketoglutarate, whereas coupled transamination ( 2 2 )
results in the formation of oxaloacetate.
It is not likely that these reactions would lead to any net increase of the pool of T C A
cycle-intermediates during exercise. Meyer & Terjung ( I 979) noted an increase in
pyruvate and alanine and a decrease in glutamate of exercising rat muscle. Since the
aminotransferases operate close to equilibrium, these changes would be accompanied
by a decrease (and not increase) of alpha-ketoglutarate and oxaloacetate during the
period of increased energy demand. In the studies of Goodman & Lowenstein ( I 977)
and Aragon & Lowenstein ( I 980), aspartate was unchanged whereas pyruvate and
alanine were slightly increased and glutamate showed a small decrease. Such changes
would be accompanied by a decrease of alpha-ketoglutarate and no change of
oxaloacetate if the transaminases maintain equilibrium. T h e increase of the pool size of
TCA-cycle intermediates which occurs during muscular work therefore cannot be due
to the action of the aminotransferases.
(ii) Pyruvate carboxylase (E.C. 6 . 4 . I . I , reaction 2 3 ) :
pyruvate
+ A T P + CO,
--f
oxaloacetate
+ A D P + Pi.
(23)
Crabtree et al. ( I972) reported that pyruvate carboxylase of vertebrate skeletal muscle
is undetectable, whereas other authors (Bottger et al., 1969; Ballard et al., 1970; Van
den Thillart & Smit, 1984) measured a small enzyme activity. Spydevold et al. (1976)
and Davis et al. ( I 980) described how intact mitochondria isolated from rat muscle are
able to carboxylate pyruvate at a rate of at least 2.5 nmole/mg protein .min at 37 O in the
ARENVAN WAARDE
282
presence of acetyl-coA and malate as a trapping pool. Oxygen uptake by mitochondria
respiring on pyruvate is stimulated 3-4 fold by bicarbonate, this stimulation being
dependent on ATP. T h e authors therefore concluded that the pyruvate carboxylase
pathway may function in the repletion of citric acid cycle intermediates which is
observed when the acetyl coA-concentration is elevated by infusion of fatty acids. It is
not clear, however, if pyruvate carboxylase has sufficient activity to contribute
significantly to the generation of Krebs-cycle intermediates during exercise. Part of the
carboxylase activity in the mitochondria1 preparation may also be due to contamination
with adipose tissue. Fat cells can be present around skeletal muscle and they possess a
high activity of pyruvate carboxylase (Ballard et al., 1970;Aragon & Lowenstein, 1980;
Aragon et al., 1981).
(iii) Cytosolic NADP-dependent malic enzyme (E.C. I . I . I .40,reaction 24):
pyruvate
+ CO, + NADPH + H+ + malate + NADP+.
(24)
When rat skeletal muscle mitochondria are incubated with pyruvate, bicarbonate and
NADPH, cytoplasmatic malic enzyme can replace added malate in stimulating
oxidation of acetyl-coA formed from pyruvate by oxidative decarboxylation
(Swierczynski, I 980).
(iv) Glutamate dehydrogenase (E.C. I .4.I . 2, reaction I I):
+
+
glutamate NAD(P)+ + alpha-ketoglutarate NAD(P)H
+ NH,+.
(1
1)
However, activity of glutamate dehydrogenase in the direction of oxidative deamination
is virtually undetectable in rat muscle (Aragon & Lowenstein, 1980;Lowenstein,
1972).
Although pyruvate carboxylase and malic enzyme may supply part of the carboxylic
acids necessary for the increase of Krebs-cycle flux during exercise, the P N C has a
greater activity (Aragon et al., 1980,1981; Scislowski et al., 1982)and a larger free
energy decline (Lehninger, 1970;Lowenstein, 1972).It is therefore not surprising that
disruption of the P N C leads to impairment of aerobic energy generation (Flanagan
et al., 1986).
Katz et al. (I 986) made human subjects perform voluntary leg exercise until fatigue
occurred. During contraction, the muscle was kept under ischaemic conditions to avoid
loss of ammonia to the circulation and to prevent aerobic phosphocreatine synthesis.
T h e activity of AMP-deaminase was evaluated from the rate of decrease of the
adenylate pool size, as determined in biopsy samples (IMP-reamination was negligible
under these experimental conditions).
AMP-deaminase activity proved to be related to the fibre composition of the muscle,
fast-twitch fibres having the highest amount of enzyme ( Y = 0.92, P < 0.001).T h e
activity of the deaminase was not related to fibre lactate content ( Y = 0.27,P > o*o5),
but positively correlated with the turnover rate of A T P as evaluated from the decline
in A T P and PCr and increase in lactate and pyruvate contents ( Y = 0.75,P < 0.001).
Ammoniagenesis was related to the decline of the size of the adenylate pool ( Y = 0.81,
P < 0.001).
T h e authors concluded that :
( a ) Muscle acidosis is not essential for the activation of AMP deaminase, since there
is no relationship between enzyme activity and lactate content. This conclusion is
The purine nucleotide cycle
283
further substantiated by the observation that iodo-acetate poisoned rat muscle shows no
change in pH during contraction, although the adenylate pool is almost completely
converted to I M P (Dudley & Terjung, 19856). In both fast-twitch red and fast-twitch
white fibres, a large increase in lactate content can under certain conditions be
accompanied by lack of AMP deamination (Dudley & Terjung, 1985b). Large
decreases in p H and activation of AMP-deaminase are therefore to a certain extent
independent events.
( b ) AMP-deaminase seems to be activated during a temporary imbalance between
ATP-consumption and ATP-production, which occurs when a high rate of ATPturnover is coupled to a low phosphocreatine level. Such a situation will lead to an
increase of free AMP and free ADP in the sarcoplasm, with concomitant activation of
the enzyme (Katz et al., 1986; see also Dudley & Terjung, 1985b). T h e greater the
capacity of the muscle for aerobic energy production, the less likely is energy shortage
and activation of AMP-deaminase to occur (Dudley & Terjung, 1 9 8 5 ~ )Training
.
of
muscles leads to an increased blood supply and increases of mitochondria1 content.
Trained muscles therefore show a less pronounced decrease of creatine phosphate, less
lactate production and a delayed activation of AMP-deaminase during exercise (Dudley
8z Terjung, 1 9 8 5 ~ ) Elimination
.
of the blood flow on the contrary leads to a more
marked decrease of creatine phosphate, elevated levels of lactate and a more rapid
activation of AMP-deaminase (Dudley & Terjung, 1985a ) .
( c ) Ammonia does not play an important role in the activation of glycolysis during
muscular work. In human muscle, exhaustive exercise is accompanied by accumulation
of ammonia to 2.3 ymollg dry weight. In the same experiment, however, anaerobic
glycolysis gave rise to the formation of 75.6 ymol hydrogen ions/g dry weight of
muscle. Thus, ammonia can buffer only 3 % of the hydrogen ions formed during
muscular contraction (Katz et al., I 986). Ammonia activation of phosphofructokinase
can be demonstrated under certain in vitro conditions, but ammonia loses its activating
effect in the presence of physiological amounts of K+ (Sugden & Newsholme, 1975).
Detailed observation of the changes in ammonia and lactate contents of muscle during
exercise suggest that there is no direct correlation between ammonia level and glycolytic
rate. In human muscle, glycolysis is activated almost instantaneously after the onset of
contraction, whereas the glycolytic rate remains constant for about 50 s. Yet, during the
first 25 s no ammonia accumulation occurs, ammoniagenesis starting only 30 s after the
beginning of the exercise (Katz et al., 1986). A similar lack of correlation in time
between ammonia production and activation of glycolysis has been observed in rat
muscle by Dudley & Terjung ( 1 9 8 5 ~ ) .During recovery of human muscle, ammonia
remains elevated for at least four min, although glycolysis is switched off immediately
after the termination of exercise (Katz et al., 1986). It is evident that other control
mechanisms, like phosphorylation and nucleotide activation of the key enzymes
phosphorylase and phosphofructokinase overrule any stimulating effect of ammonia.
Under extraordinary conditions (phosphorylase kinase-deficiency), however, AMPdeaminase may be involved in the control of glycolytic rate via IMP-activation of
phosphorylase b (Rahim et al., 1976, 1980; Aragon et al., 1980).
ARENVAN WAARDE
284
Table 4. Distribution of AMP-deaminase in tissues of invertebrates
Activity present
Activity absent o r very low
Annelida :
Abarenicola pacifica (gut)
A . oagabunda (gut)
Amphitrite robusta (gut)
Arenicola cristata
(body wall muscle)
Lumbricus terrestris
(gut and body wall)
Nereis branti (gut)
Srhizobranchia insignis (gut)
Cephalopoda :
‘Squid’ (mantle muscle)
Loligo pealii (mantle)
Crustacea :
Cheras destructor (tail muscle)
J a w s llandi (tail muscle)
Insecta :
Gastromargus musicus
(leg and thorax muscle)
Locusto migratoria
(leg and thorax muscle)
Periplaneta americana
(thorax muscle)
Crustacea :
‘Crab’ (claw muscle)
Cancer magister
Carcinus afinis (claw muscle)
Chionoecetes opilio (leg muscle)
Homarus americanus (tail muscle)
Orconectes limosus (tail muscle)
Paralithodes camtschatica
Rhithropanopeus harrisi (claw)
Pelecypoda :
Chlamys opercularis
(adductor muscle)
Crassostrea nippona
(midgut gland)
Pecten yessoensis
(adductor muscle)
Placopecten magellanicus
(adductor muscle)
Platinopecten caurinus
(adductor muscle)
Unio pictorum (adductor)
Gastropoda :
Helix aspersa (hepatopancreas)
H . pomatia (foot muscle)
Other mollusca :
Haliotis sp.
[ I ] Bishop & Barnes (1970); [z] Gibbs & Bishop (1977); [3] Umiastowski (1964); [4] MacDonnell &
Tillinghast (1973); [5] Conway & Cooke (1939); [6] Hiltz & Bishop (1975); [7] Dingle & Hines (1974); [8]
Campbell &Vorhaben (1979); [9] Saitoetal. (1958); [IO]Storey & Storey (1978); [ I I ] ChandrasenaSz Hird (1978);
[ I Z ] Gilmour & Calaby (1953); [13] Cochran (1961); [I+] Grieshaber (1978); [IS] Ishida et al. (1969); [16]
Kitagawa & Tonomura (1957); [17] Hiltz & Dyer (1970); [IS] Groninger & Brandt (1969); [19] Groninger &
Brandt (1970); [ 2 0 ] Stankiewicz (1982).
Table 5. Changes in muscle adenine nucleotide levels during exercise
(All values in pmollg wet weight, except those indicated by ‘, which are in pmol/g dry weight. AEC =
Adenylate energy charge (value without dimension); T A N = total level of adenine nucleotides. T h e upper line
for a particular species indicates the control condition; the lower line is the exercised condition. Significant
differences between control and exercised condition are indicated by asterisks (*).)
Species
Annelida
Arenicola marina
Control
1 2 h exercise
Tubifes sp.
Control
2 h exercise
ATP
ADP
AMP
TAN
5‘14
5.16
080
068*
[I1
1417
1405
0.84
0.74*
[21°
1.06
1’94*
0’52
2.52*
1029
8.4I *
3’17
408*
0 7I
1.65~
3.56
070
AEC
Ref.*
The purine nucleotide cycle
Table 5 (cont.)
Species
Pelecypoda
Chlamys opercularis
Control
Exhausted
Placopecten magellanicus
Control
Exhausted
Cephalopoda
Loligo pealii
Control
Exhausted
Crustacea
Cherax destructor
Control
Exhausted
Crangon crangon
Control
Exhausted
Insecta
Schistocerca gregaria
3 m flight
Locusta migratoria
Control
Jumping exercise
Teleostei
Carassius auratus
Control
Exhausted
Cyprinus carpio
Control
Exhausted
Macrozoarces americanus
Control
Exhausted
Mammalia
Human
Control
Exhausted
Human
Control
Exhausted
Mouse
Control
Exhausted
Rat
Control
Exhausted
Species
ATP
ADP
AMP
TAN
AEC
5'25
083*
0.66
1.89"
006
2'74*
5'97
5'46
093
033*
8.8 I
5'65*
1.38
2.38*
0.16
0.79*
'035
8.82
092
077*
5'3
07
2.2*
0.3
2.I*
6.3
6. I
0.90
052*
7'43
4.47*
094
2.76*
0.08
8.45
8.65
0'93
0.67~
204
I1.1*
2.9
9.4*
1.8*
1.42~
2' 1
7'6*
25'4
28. I
086
056"
5.06
432*
043
006
5'55
1.10*
012*
5'54
0'95
088"
6.02
5.60
054
1.96*
0.29
0.92*
6.85
8.48
092
0.78*
3'02
0 5I
002
0'73
0IO*
3.56
2'79
0.92
I'97*
412
0.97
0.73*
007
1.87*
5.16
2.68*
089
083
2.53
1'77*
029
0.49
008
2.90
0.92
026*
252
080*
3'0
3.8*
0 2
5'17
2.9 I *
085
1.29~
012
6.99
5.'4*
0'93
0.72*
005
26.6
I 6.9*
3'7
3'5
ATP
ADP
24'3
I 9.6'
0.08*
0 2
27'5
~ 3 . 6 ~
081*
0'94
0.9 I *
6.14
435*
0.9 I
7'64
549*
097
093*
04
05*
307
20.9~
0'93
0,89*
AMP
TAN
AEC
015
003*
082*
* [ I ] Surholt, 1977; [2] Schottler, 1978; [3] Grieshaber, 1978; [4] De Zwaan et al., 1980; [5] Storey & Storey,
1978; [6] England & Baldwin, 1983; [7] O m e n & Zebe, 1983; [8] Rowan & Newsholme, 1979; [9] Cerez &
Kirsten, 1965; [IO]Van Waarde & Kesbeke, 1983; [ I I ] Driedzic & Hochachka, 1976; [12] Driedzic ef al., 1981 ;
[13] Sahlin et al., 1978; [14] Sutton et al., 1980; [ I S ] Rahim et al., 1980; [16] Goodman & Lowenstein, 1977.
ARENVAN WAARDE
286
Table 6 . Changes in the adenylate pool of muscle during severe exercise
(Calculated from the data presented in Table 5 . )
ATAN ("%)
Species
Arenicola marina
Tubifex sp.
Chlamys opercularis
Placopecten magellanicus
Loligo p e a k
Cherax destructor
Crangon crangon
Schistocerca gregaria
Locusta migratoria
Mean
Standard deviation
Difference between
vertebrates and
invertebrates
Invertebrates
04
+
- 01
- 8.5
- 14'8
- 3'2
-842
- 35'9
-6 0 4
-398
- 45'6
- 14.6
- 7.0
+
+ 106
2'1
-
0 2
+ 23.8
- 37
Carassius auratus
Cyprinus carpio
Macroaoarces americanus
Human
Human
Mouse
Rat
Mean
Standard deviation
- 29.2
- 18.3
+
Ratio
-
183
10
2'5
19
-
73
-
I
24
I1
-36.9
-546
Vertebrates
-21.6
-48.1
-3 0 0
- 19'3
-4 9 0
- 26.5
-13.1
-36.5
-31.9
-36
- 26
- 142
- 29.2
- 22.9
1'7
1'1
2.3
1 '4
1 '7
1'2
1'1
I2
I2
P<05%
Not significant
(Student's t test)
VII. INVERTEBRATE TISSUES
In invertebrate tissues, the activity of AMP-deaminase is generally much lower than
in the corresponding organs of vertebrates. When low substrate concentrations ( 0 .I 5
mM) are used in a spectrophotometric assay, the muscle enzyme is usually undetectable
(Umiastowski, 1964). At saturating AMP-levels, however, enzyme activity can be
measured in some species (Umiastowski, I 964). In other species, especially insects and
bivalves, the enzyme seems to be really lacking (see Table 4).
Many authors have described the breakdown of purine nucleotides occurring in dead
animals upon storage at o "C or at freezing temperatures. Purine catabolism is of
commercial interest, since one of the metabolites, hypoxanthine, has an unpleasant
flavour and is thus spoiling the taste of fishery products. A breakdown pattern in which
ADP, AMP, IMP, inosine and hypoxanthine show subsequent accumulation is strong
evidence for the presence of AMP-deaminase in the muscle. Such evidence has been
presented for certain Crustacea (Dingle & Hines, 1974; Fraser Hiltz & Bishop, 1975;
Groninger & Brandt, 1969, 1970; see Table 4). In other invertebrates, like squid (Saito
et al., 1958) and scallop (Fraser Hiltz & Dyer, 1970) purine degradation follows the
route :
A T P + ADP + AMP + adenosine
+ inosine + hypoxanthine.
The purine nucleotide cycle
287
Table 7. Changes of adenine nucleotide levels in tissues during anoxia
(Upper line for a particular species is the control condition, lower line is the anoxic condition.
Presentation of data similar to Table 5 . )
Organism
Coelenterata
Bunodosoma cavernata
Control
3 h anoxia
Annelida
Arenicola marina
Control
1 2h anoxia
Tubifex sp.
Control
2 h anoxia
Pelecypoda
Mytilus edulis
Control
5 d anoxia
Crustacea
Orconectes limosus
Control
16 h anoxia
Teleostei
Anguilla anguilla
Control
6 h anoxia
Carassius auratus
Control
Red muscle 1 2h anoxia
Carassius auratus
Control
White muscle 12 h anoxia
Platichthys Jesus
Control
29 h hypoxia
ATP
ADP
AMP
TAN
AEC
062
0.33"
028
042"
007
0.24~
097
0'99
078
054'
3'73
2 . 9 ~ ~
085
1.16"
0.30
0.66"
488
474
085
0.74'
3'17
408"
0.7 I
1.65"
1417
1405
0.84
0.74'
069
1.34'
052*
2'95
3.16
085
0.62"
006
036*
456
5'23
082*
0.05
2.16
1.63
090
071"
406
3'36
083
632
5'09
093
089'
5'53
480
0.9 I
0.86'
10.29
8.4 I *
2.15
1.30"
409
3'75
0 4I
1'79
1.14
0'33
034
3'22
2'44
072
0.74
5.46
417
0 8I
082
463
3.79*
077
083
1.12'
0 1I
016'
012
0.18
005
010"
0.13
018
094
088
* [ I ] Ellington, 1981; [ z ] Surholt, 1977; [3] Schottler, 1978; [4] Wijsman, 1976; [5] Gade, 1984; [6] Van
Waarde et al., 1983; [7] Van den Thillart et al., 1980; [8] Jergensen & Mustafa, 1980.
This suggests AMP-deaminase activity in the latter species is much lower than the
activity of 5'-nucleotidase.
The properties of AMP-deaminase in invertebrate muscle (when the enzyme is
present) seem different from those in muscle of vertebrates. T h e invertebrate enzyme
generally has a much lower affinity for its substrate and a much lower maximal velocity
than its vertebrate counterpart (Bishop & Barnes, 1971; Gibbs & Bishop, 1977;
MacDonnell & Tillinghast, 1973 ; Stankiewicz, 1982).As a consequence, the behaviour
of the adenylate pool during an imbalance between energy consumption and energy
production is different from that in vertebrates.
Changes in the levels of purine nucleotides during severe exercise have been reviewed
in the Tables 5 and 6. It is clear that in both vertebrates and invertebrates, exercise
results in a significant drop of the ATP-content of muscle. In vertebrates, this is
ARENVAN WAARDE
288
Table 8. Changes of purine nucleotides in muscle during anoxia
(Calculated from the data presented in Table 7.)
Species
TAN
(Yo)
Invertebrates
Bunodosoma cavernata
Arenicola marina
Tubifex sp.
Mytilus edulis
Orconectes limosus
Mean
Standard deviation
+
- 468
-21.7
- 18.3
-395
- 8.3
2'1
- 29
- 0 1
7'1
+
+I 4 7
- 27
16
+ 4
7
Vertebrates
Anguilla anguilla
Carassius auratus (red)
Carassius auratus (white)
Platichthys Jesus
Mean
Standard deviation
Difference between
vertebrates and
invertebrates
(Student's t test)
-363
- 242
- 23.6
- 18.1
-245
- 26
8
- 19
Not significant
- 17'2
- 19'5
- I 3'2
5
P<05%
accompanied by activation of AMP-deaminase and a decline of the total adenine
nucleotide pool. No such activation occurs in invertebrates, however, the decline of the
nucleotide pool being small or even absent. As a consequence, the adenylate energy
charge shows a much greater drop in invertebrate muscle during exercise.
Another metabolic situation in which imbalance between ATP-consumption and
ATP-production is likely to occur is environmental anoxia. Purine metabolism in
anoxic muscle has been reviewed in the Tables 7 and 8. A similar pattern is observed
as during exercise. In both vertebrates and invertebrates, anoxia results in a significant
drop of muscle ATP-content. In vertebrates, AMP-deaminase is activated and purine
nucleotides are broken down to I M P (values not shown in Table 7), but in invertebrates,
total adenine nucleotide content does not show any decline.
We conclude that :
( a ) muscles of many invertebrate species lack adenylate deaminase, the deaminating
enzyme from the purine nucleotide cycle;
(b) even when adenylate deaminase is present, the enzyme seems not to be activated
during an imbalance between energy production and energy consumption ;
(c) the P N C therefore does not operate in the same way as in vertebrate muscle.
VIII. CONCLUSION
T h e absence of adenylate deaminase from certain muscles indicates that deamination
of AMP is not essential to the contraction process. In the absence of the enzyme, the
functions of the purine nucleotide cycle seem to be performed by other metabolic
pathway(s).
It is interesting to note that insect flight muscle, an aerobic tissue with an enormous
The purine nucleotide cycle
2 89
metabolic rate (Beenakkers et al., 1984), lacks adenylate deaminase and therefore does
not possess a complete purine nucleotide cycle. In this muscle, dicarboxylic acids are
supplied to the Krebs cycle by the action of the enzyme pyruvate carboxylase. Insect
muscle contains an enormous amount of this enzyme in comparison to vertebrate
muscle (Crabtree et al., 1972; Lowenstein, 1972). Thus, the anaplerotic function of the
P N C seems to be taken over by pyruvate carboxylase. It is not known whether other
invertebrate muscles in which the P N C is lacking have a similar replacement. Adductor
muscle of bivalves shows rather high levels of phosphoenolpyruvate carboxykinase,
which may also supply intermediates to the tricarboxylic acid cycle (Crabtree et al.,
1972).
Stabilization of the adenylate ‘energy charge ’ (or phosphorylation potential) is an
established function of the P N C in the cytosol of vertebrate cells, but such stabilization
seems to be lacking in invertebrates. Some metabolic parameters (like the rate of protein
synthesis, Edwards et al., 1979) are strongly dependent on the energy charge, whereas
others (like the synthesis of lipids, Farber, 1973) show little variation over a broad range
of charge. Since the charge is strongly stabilized in vertebrates, but much less in
invertebrates, it seems reasonable to assume that some energy-consuming process is not
allowed to slow down in vertebrates whereas it can drop in invertebrates. It is tempting
to speculate that this process might be the action of ion pumps in cellular membranes.
Invertebrate membranes generally have a much lower density of ion channels and also
show the possibility of channel arrest (Hochachka & Guppy, 1987).
IX. SUMMARY
I . T h e operation of the purine nucleotide cycle, consisting of the enzymes adenylate
deaminase (E.C. 3 . 5 . 4 . 6 ) , adenylosuccinate synthetase (E.C. 6 . 3 . 4 . 4 ) and adenylosuccinate lyase (E.C. 4 . 3 . 2 . z), has been reviewed with reference to its metabolic
function in animal tissues.
2. Abundant evidence, both from in vitro and in vivo studies, suggests that the purine
nucleotide cycle serves to stabilize the adenylate ‘energy charge ’ (or ‘ phosphorylation
potential ’) in the cytoplasm of vertebrate cells during a temporary imbalance between
ATP-consumption and ATP-production. This stabilization, however, is absent or
much less efficient in tissues of invertebrates.
3. T h e hypothesis that AMP-deaminase is involved in the regulation of glycolysis is
not supported by recent work. In a variety of cell types, including skeletal muscle and
blood platelets, blocking of AMP-deaminase activity (due to a genetic defect or to
pharmacological inhibition) is without effect on the glycolytic rate. Detailed kinetic and
histochemical analysis of energy metabolism shows lack of correlation between AMPdeaminase activity and glycolysis in skeletal muscle during exercise.
4. T h e purine nucleotide cycle appears to control the level of citric acid cycle
intermediates in skeletal muscle. Pharmacological inhibition of adenylosuccinate lyase
or adenylosuccinate synthetase leads to a reduced availability of four-carbon ‘ sparker ’
molecules to the Krebs cycle with a concomitant impairment of aerobic energy
production during muscular work.
5 . T h e cycle appears to be a major pathway for amino acid deamination in skeletal
muscle and brain of vertebrates, but not in kidney or liver.
290
ARENVAN WAARDE
X. ACKNOWLEDGEMENTS
Research by A. v. W. described in this paper was supported by the Foundation for Biological Research (BION)
which is subsidized b y the Netherlands Organisation for the Advancement of Science (ZWO). During the period
in which this article was written, the author was the recipient of a ZWO-stipend which enabled a one-year stay at
Yale University (Department of Molecular Biophysics and Biochemistry). The help of Professor A. D. F. Addink
in reading the manuscript is gratefully acknowledged.
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XII. A D D E N D U M
Since the original submission of this manuscript, two papers have been published
which deal with the difference in adenylate catabolism between vertebrates and
invertebrates. Fijisawa & Yoshino ( I 987) examined the activities of AMP-deaminase,
298
ARENVAN WAARDE
5’-nucleotidase and adenosine deaminase in muscles from vertebrates and invertebrates
at saturating substrate concentrations. They confirmed the result quoted in Table 4,
indicating that AMP-deaminase is undetectable in muscle of Haliotis gigantea. T h e
enzyme is also lacking in the cuttlefish Sepia subculeata, but present in low amounts in
muscle from the Crustacea Pandalus borealis, Paracus japonicus and Neptunus
trituberculosis, the Mollusca Scapharca broughtonii, Rapona thomasiana, Tegillarca
granosa, Batillus cornulus, Ampullaria sp. and Schizithaerus keenae, and the Cephalopoda Doryteuthis kensaki, Todarodus paci$cus, Watasenia scintillans and Doryteuthis
bleekeri. Raffin & Thebault ( I 987) purified an AMP-deaminase from the tail muscle of
the Crustacean Palaemon serratus. T h e enzyme was shown to be very different from that
in vertebrate muscle; both its specific activity and its affinity for AMP were very low.
Thus, the essential difference between vertebrates and invertebrates in regards to the
regulation of the PNC has been observed by many authors.
FIJISAWA,
K . & YOSHINO,
M . (1987). Activities of adenylate-degrading enzymes in muscle from vertebrates and
invertebrates. Comparative Biochemistry and Physiology 86 B, 109- I I 2.
RAFFIN,J. P. & THEBAULT,
M. T. (1987). Purification and partial characterization of an AMP deaminase from the
marine invertebrate Palaemon serratus. Comparative Biochemistry and Physiology 88 B, 107 1 I 076.
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