The purine nucleotide cycle and its molecular defects

Progress in NeurobiologyVol. 3g, pp. 547 to 561, 1992 Printed in Great Britain.All rights reserved

0301-0082/92/$15.00 © 1992Pergamon Press Ltd

THE PURINE NUCLEOTIDE CYCLE A N D ITS MOLECULAR DEFECTS G. VAN DEN BERGHE, F. BONTEMPS, M. F. VINCENT a n d F. VAN DEN BERGH Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology, UCL 7539, B-1200 Brussels, Belgium (Received 17 January 1992)

CONTENTS 1. Introduction 2. Enzymes of the purine nucleotide cycle 2.1. Adenylosuccinate synthetase 2.2. Adenylosuccinate lyase 2.3. AMP deaminase 3. Connections of the pufine nucleotide cycle 3.1. Connections with purine metabolism 3.2. Connections with amino acid metabolism and the Krebs cycle 4. Operation and role of the purine nucleotide cycle 4.1. Muscle 4.2. Liver 4.3. Kidney 4.4. Brain 5. Molecular defects of the purine nucleotide cycle 5.1. Muscle AMP deaminase deficiency 5.1.1. Clinical picture and diagnosis 5.1.2. Biochemical and pathophysiological aspects 5.1.3. Genetics 5.1.4. Treatment 5.2. Erythrocyte AMP deaminase deficiency 5.3. Regulatory mutations of liver AMP deaminase 5.4. Adenylosuocinate lyase deficiency 5.4.1. Clinical picture and diagnosis 5.4.2. Biochemical and pathophysiological aspects 5.4.3. Genetics 5.4.4. Treatment 6. Summary and conclusions Acknowledgements References

I. INTRODUCTION In the late 1920s, J. K. Parnas and W. Mozolowski (1927) in Poland, and G. Embden and coworkers (1928) in Germany, showed that muscle contraction is accompanied by production of ammonia and by conversion of A M P into IMP. The formation of ammonia was equivalent to the conversion of adenine nucleotides into IMP under anaerobic conditions, but markedly surpassed this conversion in the presence of oxygen. This led Parnas to the farsighted hypothesis that amino acids are used for the aerobic regeneration of A M P (reviewed by Van Waarcle, 1988). Although adenylate deaminase, the enzyme responsible for the production of ammonia from AMP, was simultaneously discovered (Schmidt, 1928), nearly 30 years elapsed before the two enzymes involved in the regeneration of AMP, adenylosuccinate synthetase (Abrams and Bentley, 1955) and adenylosuccinate lyase (Carter and Cohen, 1955), were identified.

547 547 548 548 549 550 550 551 551 551 552 553 553 554 554 554 554 555 555 555 555 556 556 556 557 557 557 557 557

Sixteen years later, J. M. Lowenstein and K. Tornhelm (1971) formulated the concept that the three enzymes formed together a functional unit (Fig. 1), which they termed the "purine nucleotide cycle". In this article, the main properties of the enzymes of the purine nucleotide cycle, its connections with other pathways, operation and proposed role in different tissues, and molecular defects in man will be reviewed.

2. ENZYMES O F THE PURINE NUCLEOTIDE CYCLE The three enzymes of the purine nucleotide cycle have been found in nearly all organisms and tissues examined. A major exception is the human erythrocyte, which does not contain adenylosuccinate synthetase and consequently cannot form adenine 547

548

G. VANDEN BERGHEet al. COO "

I

H--C--C

I

H2-COO-

N

I

Hbose

5'-P

adenylosuccinate

adeny/or,uccinate lyaso

synthetase

AS

IMP '

\7"

{

AMP

NH3 0

N H2

I

I

rlbole 5'-P

rlbose 5 ' - P AMP ~ a m i ~ , e

FIG. I. The purine nucleotide cycle. AS, adenylosuccinate; ASP, aspartate; FUM, fumarate; Pi, inorganic phosphate. nucleotides from IMP (Lowy et al., 1962). A brief review of the main properties of adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminase, will be restricted to the mammalian enzymes. 2.1. ADENYLOSUCCINATESYNTHETASE Adenylosuccinate synthetase [EC 6.3.4.4.] catalyzes the condensation of IMP and aspartate into adenylosuccinat¢. The reaction requires GTP which is hydrolyzed into GDP and Pi for the formation of an activated intermediate (reviewed by Stayton et al., 1983). Adenylosuccinate synthetase has a MW of about 100,000 and is composed of 2 subunits. The enzyme exists under two isoforms: type M, a basic protein with an isoelectric point of 8.9, and type L, an acidic protein with an isoelectric point of 5.9 (Matsuda et al., 1977). Skeletal and heart muscle contain only type M, liver contains both type M and L in near equal amounts, and brain and kidney contain predominantly type L. The relative percentage of the acidic isozyme increases in regenerating liver (Matsuda et al., 1978), in the liver of animals fed a purine-free diet (Baugher et al., 1980) and in various tumor cells (Ikegami et al., 1989). Adenylosuccinate synthetase displays hyperbolic kinetics with its three substrates. The values of the Km of IMP (200-700/aM) for type M adenylosuccinate synthetase measured by various authors (reviewed by Stayton et al., 1983) are higher than the concentration of IMP in resting muscle (about 70pM). Km of IMP for the L form is reportedly lower than for the M form, but is still higher than the basal concentration of IMP in liver (less than 10/aM). In contrast, for both isoforms of adenylosuccinate synthetase, the Km values of aspartate (around 300pM) and GTP (10--20/aM) are lower than their respective tissue

concentrations (600-800#M for aspartate, about 500/aM for GTP). Taken together, these data indicate that the concentration of IMP determines the activity of the enzyme whereas aspartate and GTP are always saturating. The products of adenylosuccinate synthetase are fairly strong inhibitors of the reaction. with K, in the 5-50/aM range for adenylosuccinate and GDP, and of about 25 mM for Pi. GMP, ADP and AMP are also inhibitory (Stayton et al., 1983). Studies of the influence of ADP and AMP on the activity of adenylosuccinate synthetase in crude skeletal muscle extracts show that, notwithstanding the multiplicity of the inhibitory effects exerted on the enzyme, its intracellular activity seems mainly determined by the concentration of IMP (Manfredi and Holmes, 1984). This may be explained by extensive protein binding of the inhibitory nucleotides. Inhibition, particularly of type M adenylosuccinate synthetase by fructose-i,6-bisphosphate has been reported and proposed to play a role in the synchronization of glycolysis with the purine nucleotide cycle (Ogawa et al., 1976). This is, however, unlikely in view of the high K, of fructose-l,6-bisphosphate as compared with its physiological concentration in muscle. Two potent inhibitors of adenylosuccinate synthetase, both analogs ot" its substrates, have been identified. Hadacidin, a natural antibiotic which resembles aspartate, competes with the latter (Shigeura and Gordon, 1962). Alanosinc, also a bacterial product, is conjugated by SAICAR (succinylaminoimidazole carboxamide ribotide) synthetase. the 7th enzyme of de novo purine synthesis, into alanosyl-CAIR (carboxyl-aminoimidazole ribotide), which competes with IMP (Tyagi and Cooney, 1980). 6-Mercaptopurine, an hypoxanthine analog which inhibits several enzymes of purine metabolism owing to its conversion by hypoxanthine-guanine phosphoribosyltransferase into the AMP analog 6-mercaptopurine ribotide, also inhibits adenylosuccinate synthetase (Clark and Rudolph, 1976). The gene of adenylosuccinate synthetase has been localized on chromosome I in humans (Lai et al., 1989). Recently, a mouse muscle eDNA that encodes the basic isozyme (Guicherit et al., 1991), and a human liver eDNA (Powell et al., 1991), of which the predicted amino acid sequence shows 70% identity with that of mouse muscle adenylosuccinate synthetase, have been cloned. The cDNAs encode, respectively, a 452 and 455 amino acid sequence, corresponding to a subunit MW of about 50,000. 2.2. ADENYLOSUCCINATE LYASE

Adenylosuccinate lyase (EC 4.3.2.2, also named adenylosuccinase) cleaves the C-N bond of adenylosuccinate, yielding AMP and fumarate. It also catalyzes the cleavage of SAICAR into AICAR and fumarate, the 8th step of the de novo pathway of purine synthesis. Both reactions are similar to that catalyzed by the urea cycle enzyme, argininosuceinate lyase (reviewed by Ratner. 1972). Studies of purified rat muscle adenylosuccinate lyase have shown that the enzyme has a MW of approx. 200,000 and is composed of 4 subunits (Casey and Lowenstein 1987a). Adenylosuccinate lyase has also

PURINE NUCLEOTIDECYCLE

been partially purified from rat liver (Smith et al., 1982) and from human erythrocytes (Barnes and Bishop, 1975). That isoforms of adenylosuccinate lyase exist is apparent from two observations. Firstly, whereas starvation induced a profound decrease of the activity of adenylosuccinate lyase in rat liver and spleen, it had no effect on the activity of the enzyme in muscle, brain and kidney (Brand and Lowenstein, 1978a). Secondly, studies in patients with adenylosuccinate lyase deficiency (see Section 5.4.2) show that the activity of the enzyme is lost to a different extent in various tissues, and is normal in others. The isozymes of adenylosuccinate lyase have, however, not yet been characterized. Adenylosuccinate lyase displays hyperbolic kinetics. K~ of both adenylosuccinate and SAICAR is I-3/~M (Barnes and Bishop, 1975; Casey and Lowenstein, 1987a), in keeping with the very low intracellular concentrations of the intermediates of purine synthesis. Adenylosuccinate lyase is inhibited by the products of the two reactions which it catalyzes. K, is about 6/aM for AMP, 5-10/.tM for AICAR, and 160pM for fumarate (Barnes and Bishop, 1975; Sabina et al., 1982). Intraperitoneal administration of AICAriboside, which is phosphorylated into AICAR by adenosine kinase (Sabina et al., 1985; Vincent et al., 1991) has been used to inhibit adenylosuccinate lyase in skeletal muscle of rats in vivo (Flanagan et al., 1986). Adenylophosphonopropionate, in which the aspartate moiety of adenylosuccinate is replaced by 3-phosphoalanine, is the most potent inhibitor (K, ~ 0.02 #u) of the enzyme reported sofar (Brand and Lowenstein, 1978b). The fluorinated derivatives of aspartate, threo-[3-fluoroaspartate and erythro-[3-fluoroaspartate, also potently inhibit adenylosuccinate lyase after their conversion into adenylosuccinate and SAICAR derivatives by adenylosuccinate synthetase and SAICAR synthetase, respectively (Casey et al., 1986; Casey and Lowenstein, 1987a). The adenylosuccinate synthetase inhibitor, alanosyl-CAIR, also inhibits adenylosuccinate lyase (Casey and Lowenstein, 1987b). The gene of human adenylosuccinate lyase has been localized on chromosome 22 (Van Keuren et al., 1987). Its eDNA has been cloned in chicken liver (Aimi et al., 1990) and more recently in human liver (Stone et al., 1991). It encodes a 459 amino acid sequence, predicting a subunit with a MW of about 50,000. The genes of chicken and human adenylosuccinate lyase, and of human argininosuccinate lyase, share regions of extensive homology. 2.3. AMP DEAMINASE AMP deaminase [EC 3.5.4.6] hydrolyzes AMP into IMP and NH 3 (reviewed by Zielke and Suelter, 1971a). It has a MW of approx. 320,000 and is composed of 4 subunits. Initially reported lower molecular weights seem to result from proteolyis during purification (Marquetant et al., 1989). Several isoforms of AMP deaminase have been identified. In adult rat, three AMP deaminase isozymes are found. Skeletal muscle contains nearly exclusively isozyme A, liver and kidney a major isozyme labelled B, and

549

heart mainly isozyme C. Brain, lung and spleen contain isozymes B and C, and three hybrids formed by combination of these isoforms (Ogasawara et al., 1975a; 1978). The pattern of the rat AMP deaminase isoforms displays changes during development. Embryonic muscle contains isozyme B, which is replaced at birth by isoform A (Marquetant et al., 1987). In brain, the relative proportions of isoforms B and C and of their hybrids change markedly over the 30 days following birth (Ogasawara et al., 1975b). In humans, four AMP deaminase isozymes have been identified, which have been termed M, L, El and E2 (Ogasawara et al., 1982). lsozyme M is found in muscle, and isozyme L in liver and brain. Erythrocytes contain a major isozyme, El, and a minor isoform E2. Heart, kidney and spleen contain isozymes L, El and E2. Mononuclear blood cells and platelets contain only isozyme L and granulocytes only E2 (Ogasawara et al., 1984a). Chromatographic and kinetic analyses have shown that a shift from a fetal to an adult isoform also occurs in human skeletal muscle (Kaletha et al., 1987; Kaletha and Nowak, 1988). AMP deaminase displays complex, allosteric kinetic properties. In the absence of effectors, the substrate kinetics of brain (Setlow and Lowenstein, 1967, 1968; Ito et al., 1988), muscle (Smiley and Suelter, 1967), erythrocyte (Lian and Harkness, 1974), and liver (Van den Berghe et al., 1977a) AMP deaminase are sigmoidal. Nevertheless, S05 of AMP and n H vary considerably depending on tissue, isozyme and species. Substrate saturation curves are markedly sigmoidal with rat isozymes A and B, but nearly hyperbolic with isoform C (Ogasawara et al., 1975a). The human isozymes M and El display hyperbolic kinetics with Km of AMP of 0.6 raM, whereas isoform L has sigmoid kinetics with So 5of 6.6 mu (Ogasawara et al., 1982; Kaletha and Nowak, 1988). AMP deaminase is as a rule stimulated by ATP and ADP at concentrations in the physiological range, and by monovalent alkali metal cations such as K ÷ (Setlow and Lowenstein, 1967, 1968; Lian and Harkness, 1974; Wheeler and Lowenstein, 1979). These stimulators decrease S05 and can abolish the sigmoidicity of the substrate saturation curve, but do not influence Vma~. The potency of the stimulators varies, depending on the isozyme: ADP strongly stimulates the muscle form (Ronca-Testoni et al., 1970), but has little effect on the liver and brain enzymes. AMP deaminase is usually inhibited by physiological concentrations of GTP (Setiow and Lowenstein, 1968; Tomozawa and Wolfenden, 1970; Van den Berghe et al., 1977a) and Pi (Lee and Wang, 1968; Wheeler and Lowenstein, 1979). Both increase S0.5 and accentuate sigmoidicity of the substrate saturation curve, without influencing Vm~. Erythrocyte AMP deaminase is, however, not influenced by GTP (Lian and Harkness, 1974). The activity of AMP deaminase is also influenced by other nucleoside di- and triphosphates and by a variety of metabolites, among which phosphate esters such as glycerate-2,3-bisphosphate (Lian and Harkness, 1974), polyamines (Yoshino et al., 1978), and fatty acids (Yoshino et al., 1979). Muscle AMP deaminase can bind to myosin, an interaction which increases the activity of the enzyme by decreasing its sensitivity to inhibition by GTP

550

G. VAN DEN BERGHE et al.

(Ashby and Frieden, 1978; Barshop and Frieden, 1984). A number of A M P analogs inhibit AMP deaminase, among them 3-fl-D-ribofuranosyladenine 5'phosphate, in which the ribose is attached to the 3-instead of the 9-position of adenine (Setlow and Lowenstein, 1968; Zielke and Suelter, 1971b). The tight-binding adenosine deaminase inhibitors, coformycin and to a lesser extent 2'-deoxycoformycin. although at higher concentrations than those required to inhibit the latter enzyme, are also inhibitors of AMP deaminasc (Agarwal and Parks, 1977; Van den Berghe et al., 1980). The nucleotide, 2'deoxycoformycin 5'-phosphate, is an even more potent inhibitor of muscle AMP deaminase than its parent nucleoside (Frieden et al., 1979). These findings may be explained by the recently deduced close structural homologies of the catalytic sites of adenosine deaminase and AMP deaminase (Chang et al., 1991). Recent work has shown that mammalian AMP deaminase is encoded by a multigene family (Sabina et al., 1989a, 1990; Morisaki et al., 1990; BauschJurken et al., 1991). Three different genes for AMP deaminase have been identified. AMPD I is located on chromosome I and encodes isozymes A in rat and isoform M in man. The rat A M P D I gene spans approximately 21 Kb, contains 16 exons and produces two 2.5 Kb transcripts, which encode peptides with MW of 80,000 and 77,500. One transcript retains the very small exon 2, composed of 12 bp, which is removed in the other. The significance of this alternative splicing for which developmental and tissuespecific patterns are observed (Sabina et al., 1989a; Mineo et al.. 1990) is unknown. In humans, the A M P D 1 gene spans approximately 23 Kb, also contains 16 cxons, and produces a single 2.5 Kb transcript. Alternative splicing has not been detected. AMPD 1 is expressed at highest levels in adult skeletal muscle and at considerably lower levels in adult cardiac and embryonic skeletal muscle. A M P D 2 is also located on chromosome I and encodes isoform B in rat and isoform L in humans (Morisaki et al., 1990). It spans approximately 8 Kb and produces a single 3.4Kb transcript. A M P D 2 is expressed at highest levels in liver and brain. It is also expressed in adult cardiac muscle, which contains nearly equal amounts of AMPD 1 and AMPD 2 transcripts. AMPD 2 is also highly expressed in embryonic muscle, but almost undetectable in adult skeletal muscle. A M P D 3 has been putatively assigned to chromosome I1, spans approximately 40 Kb, and encodes isoform E I (Bausch-Jurken et al., 1991).

3.1. CONNECrlONS WITH PURINE METABOLISM

IMP, the substrate of adenylosuccinate synthetase and the product of AMP deaminase, is also formed by the de not'o pathway of purinc biosynthesis (Fig. 2). This pathway leads from phosphoribosyl pyrophosphate to IMP via 10 steps, of which the 8th is also catalyzed by adenylosuccinate lyase. Besides conversion into AMP and the other adenine nucleotides, IMP can bc transformed into GMP and the other guanine nucleotides via XMP. or degraded into inosine by cytosolic 5'-nucleotidase(s). Purinc nucleoside phosphorylasc phosphorolyzes inosine into ribose l-phosphate and hypoxanthine, the tcrminal product of purinc catabolism in tissues which do not possess xanthinc dehydrogenase. In cell types that contain the latter enzyme, hypoxanthinc is further catabolized into xanthine and uric acid (not shown in Fig. 2). Uric acid is the poorly soluble terminal product of purine catabolism in man and higher apes. In other mammals, an additional enzyme, uricase. converts uric acid into much more soluble allantoin. AMP, the product of adenylosuccinate lyase and the substrate of AMP deaminase, is maintained in equilibrium with ATP and ADP by adenylate kinase. It can also be dephosphorylated into adenosine by cytosolic 5'-nucleotidase(s). Adenosine can enter the purine catabolic pathway by deamination into inosine by adenosine deaminase. It is, however, preferentially rephosphorylated into AMP by adenosine kinasc, owing to the lower K~ of adenosine for the latter enzyme as compared with adenosine deaminasc (Arch and Newsholmc, 1978). The purine salvage enzymes, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferasc (not shown in Fig. 2) can also form. respectively. IMP and GMP, and AMP, from the corresponding purine bases. One should be aware of the fact that, because of the high activity of adenylate kinase and the necessity to maintain ATP, the energy currency of the cell, the catabolism of AMP should be strictly controlled. This is achieved in part by the low activity of cytosolic 5'-nucleotidase(s) toward AMP (Van den Berghe PRPP "~ novo ~ i

SAICAR (2) , ~ FUM AICAR

GMP

IMP ~ {4) ~,~ PI Ino q HX

3. CONNECTIONS OF THE PURINE NUCLEOTIDE CYCLE

Because adenylosuccinate synthetase, adenylosuccinate lyase and AMP deaminase share substrates and products with other enzymes, the purine nucleotide cycle is connected with other pathways of purine and of intermediary metabolism. Knowledge of these connections is essential for evaluation of the role of the purine nucleotide cycle.

AS

N ~(3)

AMP : ~ (4) PI {8) A~dd3 (., o

: 2 AOP

NH3

FIG. 2. Connections of the purine nucleotide cycle with purine metabolism. Ado, adenosine; AICAR, aminoimidazole carboxamide ribotide; AS, adenylosuccinate; FUM, fumarate; HX, hypoxanthine; Ino, inosine; Pi, inorganic phosphate; PRPP, phosphoribosyl pyrophosphate; SAICAR, succinylaminoimidazole carboxamide ribotide. (I) adenylosuccinate synthetase; (2) adenylosuecinate lyas¢; (3) AMP deaminase; (4) eytosolic 5'-nucleotidase; (5) purine nucleoside phosphorylase; (6) adenylate kinase; (7) adenosine deaminase; (8) adenosine kinase.

])URINE NUCLEOTIDECYCLE

et al., 1977b, 1988; Bontemps et al., 1988; Truong et al., 1988), and/or by recycling of adenosine by adenosine kinase (Bontemps et al., 1983). However,

owing to the much higher activity of cytosolic 5'nucleotidase toward IMP, the preservation of AMP requires in addition, either a profound inhibition of AMP deaminase, or if AMP deaminase is active, a commensurate rate of reconversion of IMP into AMP. Kinetic studies of rat liver AMP deaminase have shown that at the concentrations of its substrate and effectors prevailing in the liver under physiological conditions, namely 0.2 mM or less for AMP, 3 mM for ATP, 0.5mM for GTP and 5ram for Pi, the activity of the enzyme is more than 95% inhibited (Van den Berghe et al., 1977a). Experiments performed with human red blood cell AMP deaminase have similarly shown that the enzyme is nearly inactive at the concentrations of its substrate and effectors prevailing in the erythrocyte, namely 10/~M for AMP, i mM for ATP, I mM for Pi, and 3 mM for unbound 2,3-bisphosphoglycerate (Bontemps and Van den Berghe, 1989). These studies accord with the proposal that, at least in certain cell types, inhibition of AMP deaminase plays a crucial role in the preservation of the cellular adenine nucleotides. As will be discussed in Section 4.1, muscle AMP deaminase is most likely markedly inhibited under resting conditions, but becomes active during intense exercise. 3.2. CONNECTIONS WITH AMINO ACID METABOLISM AND THE KREBS CYCLE

The purine nucleotide cycle is linked to amino acid metabolism by glutamate-oxaloacetate transaminase, which converts glutamate into aspartate (Fig. 3). Glutamate can be formed from glutamine by glutaminase; it can also be converted into ct-ketoglutarate and ammonia by glutamate dehydrogenase. The activities of glutaminase and glutamate dehydrogenase are high in liver and kidney, lower in brain, and exceptionally low in muscle (reviewed in Lowenstein, 1972; Lowenstein and Goodman, 1978). The purine nucleotide cycle is also connected with the Krebs cycle, since fumarate is an intermediate of the latter. Because the enzymes of the purine nucleotide cycle are located in the cytosol, entry of fumarate into the Krebs cycle requires its prior conversion into malate GLU-NH 2

~

MAL NH3 ,~k

OAA aKG

[

atu . ~ J : ,z* ( oJ(G v4"~ NADH NH3

, IMP

*

AS

Fo.

~) (:)) [

AMP

Krebs

CIT

/

NH3

FIG. 3. Connections of the purine nucleotide cycle with amino acid metabolism and the Krebs cycle. AS, adenylosuccinate; ASP, aspartate; CIT, citrate; FUM, fumarate; GLU, glutamate; GLU-NH2, glutamine; ctKG, a-ketoglutarate; MAL, malate; OAA, oxaloacetate; SUC, succinate. (I) adenylosuccinate synthetase; (2) adenylosuecinate lyase; (3) AMP deaminase; (4) glutamate--oxaloacetatetransaminase; (5) glutaminase; (6) glutamate dehydrogenase. JPN 391~*~ H

551

by fumarase, followed by transport of malate into the mitochondria (not detailed in Fig. 3).

4. OPERATION AND ROLE OF THE PURINE

NUCLEOTIDE CYCLE The concept of the purine nucleotide cycle raises two questions: (1) does it operate, i.e. is there a continuous or intermittent metabolic flux from AMP into IMP, accompanied by reconversion of IMP into AMP via adenylosuccinate? (2) which role(s) does this recycling fulfil? From the observations, reviewed in Sections 2.1 and 2.3, that the isoforms of adenylosuccinate synthetase and AMP deaminase vary in their kinetic and regulatory properties, it can be inferred that the function of the cycle will not be the same in all tissues. In this section, the available evidence for or against operation of the purine nucleotide cycle, and the various roles which this operation could fulfil, will be discussed. The three main roles which have been proposed for the purine nucleotide cycle are: (1) removal of AMP, in order to pull the adenylate kinase reaction in the direction of formation of ATP, thereby increasing the ATP/ADP ratio; (2) liberation of ammonia from amino acids for urea formation or maintenance of acid-base homeostasis; (3) production of Krebs cycle intermediates, leading to an enhancement of aerobic energy production. These potential functions have been most extensively investigated in muscle, but also in liver, kidney and brain (for reviews see: Lowenstein, 1972; Van Waarde, 1988). 4.1. MUSCLE Muscle contraction requires ATP to drive actomyosin ATPase. As long as exercise has not depleted the muscle energy store, creatine phosphate, ATP can be regenerated from ADP by myofibrillar creatine kinase. However, when the intensity of exercise has exhausted creatine phosphate, regeneration of ATP depends on other processes. Operation of the purine nucleotide cycle can, in theory, regenerate ATP by three mechanisms: (1) Extraction of energy from the adenylate pool. By converting AMP into IMP, operation of AMP deaminase can pull adenylate kinase in the direction: 2 ADP--,AMP + ATP (Fig. 2). This process should result in recovery of half of the amount of ADP generated by muscle contraction under the form of high-energy phosphate. However, this pull mechanism can only operate if AMP is not regenerated immediately. On the other hand, as already discussed in Section 3.1, recycling of AMP via the purine nucleotide cycle has the advantage of preventing depletion of the adenine nucleotide pool. (2) Stimulation of muscle glycolysis, thereby enhancing both the anaerobic and aerobic generation of ATP. Several factors have been proposed to stimulate glycolysis upon operation of AMP deaminase (Lowenstein, 1972). The liberation of NH 3, which in muscle is produced nearly exclusively by AMP deaminase, could exert a direct, pH-independent stimulatory effect on phosphofructokinase, the main regulatory enzyme of muscle glycolysis. Alternatively, NH3 could buffer the inhibitory effect on phospho-

552

G. VAN DEN BERGHEet al.

fructokinase of the H • ions produced by hydrolysis of ATP. However, the observation that muscle AMP deaminase-deficient patients do not form NH3, but do produce lactate normally upon muscle contraction (see Section 5.1.1) renders a role of NH~ in the stimulation of muscle glycolysis unlikely. (3) Production of Krebs cycle intermediates. By releasing fumarate, the purine nucleotide cycle would play an anaplerotic role for the citric acid cycle, resulting in an increase of its capacity to produce A T E Numerous m vitro and in t'h,o experiments have been aimed at demonstrating that the purine nucleotide cycle functions during intense muscle exercise. Studies in cell-free extracts are hampered by the necessity to reproduce conditions that mimic muscle doing work, and to avoid build-up of inhibitors of adenylosuccinate synthetase, adenylosuccinate lyase and AMP deaminase. Nevertheless, evidence of the ability of these three enzymes to operate as a cycle, successively aminating and deaminating purine nucleotides, has been provided by a series of elegant experiments with reconstituted muscle systems by Lowenstein and his coworkers (Lowenstein and Tornheim, 1971; Tornheim and Lowenstein, 1972, 1973; Lowenstein and Goodman, 1978) and by others (Manfredi and Holmes, 1984). Even more convincing evidence of the operation of the cycle has been given by the demonstration that its components, NH~, IMP, adenylosuccinate and fumarate, the latter together with its derivative malate, increase several-fold during exercise in muscle in situ (Goodman and Lowenstein, 1977; Aragon and Lowenstein, 1980). That these increases occur within a short time interval during sustained exercise indicates that the cycle functions as a whole during this process rather than, as claimed by some authors, in two phases: deamination during contraction, followed by reamination during recovery. Nevertheless, in ectothermic species such as trout, the two processes might be temporally separated (Mommsen and Hochachka, 1988). The purine nucleotide cycle has been reported to be more active in fast-twitch than in slow-twitch muscle (Meyer and Terjung, 1979; Meyer et al., 1980). This may be related to lower activity, and differences in the regulation, of AMP deaminase in the latter type of fibers (Tullson et al.. 1990). In resting muscle, AMP deaminase is poorly active, as evidenced by the low production of NH~ (approximately 2.5 nmol/min per g of muscle) under this condition (Goodman and Lowenstein, 1977). The trigger which results in the operation of the purine nucleotide cycle during sustained contraction, implicating a priming, more than ten-fold increase of the activity of AMP deaminase as indicated by an enhancement of similar magnitude of the production of NH 3, is not immediately apparent. Both an increase of its substrate AMP, and of ADP, the main stimulator of the muscle isoform of AMP deaminase, could play a major role. These increases are, however, small and often barely discernible when measuring nucleotide levels in exercising muscle (Goodman and Lowenstein, 1977; Lowenstein and Goodman, 1978). It has therefore been proposed that muscle contraction results in an increase of freely available AMP and ADP. Why, based on calculations from the equilibria ofcreatine kinase and adenylate kinase, less

than I% of total muscle AMP is freely available, remains an open question since no AMP-binding protein has been identified in muscle. ADP, however. is known to be tightly bound to muscle actin. An additional factor in the stimulation of AMP deaminase during intense muscle contraction may be the substantial increase of its binding to myosin (Shiraki et al., 1981), in response to the decrease in ATP (Marquetant et al., 1989). As mentioned in Section 2.3, this binding leads to a marked decrease of the inhibitory effect of GTP. AMP deaminase is probably also stimulated by the lowering of pH, caused by lactate accumulation (Dudley and Terjung, 1985). Taken together, the available evidence shows that the purine nucleotide cycle operates during intense muscle exercise, accounting for the accompanying marked elevation of the production of ammonia. Its main contribution to muscle function is probably the production of Krebs cycle intermediates. However, as will be discussed in Section 5.1, findings in muscle AMP deaminase deficiency indicate that operation of the purine nucleotide cycle does not seem to be required for normal muscle function. 4.2. LIVER

One of the major liver functions is the formation of urea, the end product of protein catabolism in mammals. Urea is classically considered to arise mainly from NH 3 generated by mitochondrial glutamate dehydrogenase (Fig. 3). This concept has been questioned on the basis that the in vitro activity of liver glutamate dehydrogenase should be too low to account for the in vivo rates of urea synthesis, and it has been proposed that NH 3 is produced by the purine nucleotide cycle (McGivan and Chappell, 1975; Moss and MeGivan, 1975). Subsequent experiments have, however, invalidated this theory. If NH~ was produced by the purine nucleotide cycle, inhibition of one of its enzymes should decrease ureogenesis. The production of urea by isolated rat hepatocytes was, however, not influenced by the inhibitor of adenylosuccinate synthetase, hadacidin (Rognstad, 1977). If the NH~ which is used in the formation of urea is provided by the purine nucleotide cycle, the rate of turnover of the 6-NH: group of AMP and the rate of production of ammonia must be similar. Accordingly, incorporation of ~N from amino acids into AMP and urea should proceed with comparable velocities. Studies ot" the incorporation of USN]alanine into the 6-NH2 group of hepatic adenine nucleotides have, however, shown that its initial rate reached only about 5% of that in urea (Krebs et al.. 1978). That the purine nucleotide cycle is only a very minor source of NH3 in liver is in agreement with kinetic studies of liver AMP deaminase, as already discussed in Section 3.1. These have shown that the enzyme is profoundly inhibited under physiological conditions, in keeping with the necessity to preserve the adenine nucleotide pool as a whole. This profound inhibition of A M P deaminase also accords with the requirement to strictly control the production of uric acid, the poorly soluble end product of hepatic purine catabolism in the species that lack uricase, namely man and higher apes. The import-

PURINE NUCLEOTIDECYCLE

ance of the physiological inhibition of hepatic AMP deaminase is further corroborated by the observation that release of the inhibition results in depletion of ATP. Disinhibition of AMP deaminase can be induced by a number of compounds, of which the best known is fructose, which have in common that they can be rapidly phosphorylated in the liver (Van den Berghe et al., 1980; Van den Berghe, 1986, Vincent et al., 1989). This rapid phosphorylation results in a decrease of the concentrations of Pi and GTP, the physiological inhibitors of AMP deaminase. In humans and in experimental animals, the disinhibition of hepatic AMP deaminase and the ensuing depletion of ATP are manifested by an increase of the plasma and urine concentrations of the terminal purine catabolites, uric acid or allantoin, respectively. 4.3. KIDNEY One of the major functions of the kidney is the production of NH3 which combines with H ÷ ions to maintain acid-base homeostasis. This production increases several-fold in metabolic acidosis. The main source of the NH3 is glutamine, which is extracted by the kidney and deaminated by phosphate-dependent glutaminase (Fig. 3) The resulting glutamate is generally believed to be in turn oxidatively deaminated by glutamate dehydrogenase. Lowenstein (1972) has, however, proposed that glutamate is transaminated into aspartate by glutamate-oxaloacetate transaminase, and that NH~ is subsequently produced from aspartate by the purine nucleotide cycle. With experiments analogous to those performed with muscle cell-free extracts, Lowenstein and his coworkers could show that adenylosuccinate synthetase, adenyIosuccinate lyase and AMP deaminase are also able to function as a cycle in kidney cortex (Bogusky et aL, 1976). The quantitative contribution of the purine nucleotide cycle to the renal production of NH 3 under control as well as under acidotic conditions has, however, been a subject of controversy. The activities of kidney adenylosuccinate synthetase and adenylosuccinate lyase, but not of AMP deaminase, increase in a more parallel degree with the elevation of the excretion of NH3 during acidosis than the activities of glutaminase and glutamate dehydrogenase (Bogusky et al., 1981). This has been considered suggestive for a regulatory role of the purine nucleotide cycle in renal ammoniagenesis. Against this conclusion, however, is the fact that the activities of the enzymes of the purine nucleotide cycle, both in control conditions and in acidosis, are more than an order of magnitude lower than those of glutaminase and glutamate dehydrogenase. In isolated kidney tubules incubated with an inhibitor of transaminases, the rates of production of NH 3 from aspartate were similar to those recorded from glutamate (Bogusky et al., 1983). This indicates that the purine nucleotide cycle and glutamate dehydrogenase can function with the same velocities. These results have, however, been criticized because the presence of enzyme inhibitors and of high concentrations of aspartate may not reflect the physiological situation. Comparison of the incorporation of amino acid nitrogen into the 6-NH 2 group of AMP, to that into free NH 3, has provided the best assessment of the role

553

of the purine nucleotide cycle in renal ammoniagenesis. Nissim et al. (1985, 1986) found that in renal tubules from rats in normal acid-base status, more 15N from [2-15N]glutamine, [15N]glutamate or [~SN]aspartate was incorporated into the 6-NH2 group of the adenine nucleotides than in NH3. This indicates significant entry of amino acid nitrogen into the purine nucleotide cycle under physiological conditions. Interestingly, addition of l mM AICAriboside, most likely resulting in an increase of AICAR and IMP, and consequently of adenylosuccinate synthetase activity, enhanced the incorporation of 15N from [ISN]aspartate into adenine nucleotides (Nissim et al., 1986). At 5raM, however, AICAriboside decreased incorporation, probably owing to the inhibitory effect of AICAR accumulation on adenylosuccinate lyase. In renal tubules of acidotic rats, the rates of conversion of [2-~SN]glutamine, [~SNlglutamate or [~SN]aspartate into [6-~SN]adenine nucleotides were markedly lower than the rate of the formation of ~SNH3. This demonstrates that glutamate dehydrogenase rather than glutamate transamination followed by entry of aspartate into the purine cycle is primarily responsible for the acidosis-induced enhancement of renal ammoniagenesis. This conclusion was corroborated by Tornheim et al. (1986), who also found very little incorporation of [2JSN]glutamine, [~SN]glutamate or ['SN]aspartate into adenine nucleotides of acidotic kidney tubules. Present evidence thus indicates that in normal acid-base status the activity of the purine nucleotide cycle can account for the rate of release of NH3 from glutamate, but that under acidotic conditions the increased formation of NH 3 is explained by an increased activity of glutamate dehydrogenase. 4.4. BRAIN

Cerebral tissue can produce NH 3 (Folbergrova et al., 1969) under a variety of conditions, including

electrical stimulation, administration of convulsive agents and ischemia, which have in common that they induce depletion of creatine phosphate and ATP. On the basis that the activities of brain glutaminase and glutamate dehydrogenase are too low to account for the rates of production of NH3 recorded under the conditions listed, whereas those of the enzymes of the purine nucleotide cycle are sufficient, Schultz and Lowenstein (1976) have proposed that brain NH 3 is produced by operation of the purine nucleotide cycle. In experiments with cell-free brain extracts, analogous to those performed in muscle and kidney although complicated by dephosphorylation of the components of the purine nucleotide cycle by 5'nucleotidase(s) and phosphatases, these authors were able to show successive amination of IMP and deamination of AMP. Moreover, in brain samples obtained by freeze-blowing in rats subjected to electrical shock treatment, they recorded marked elevations of NH 3, IMP, and adenylosuccinate (Schultz and Lowenstein, 1978). The time courses of these elevations were closely parallel, but the increase of NH 3 surpassed several-fold that of IMP. Although the discrepancy might be explained by reutilization of IMP by adenylosuccinate synthetase, a participation of glutamate dehydrogenase in the production of

554

G. VAN DEN BERGrlEet al.

NH~ was also considered possible. Taken together, the available data indicate that in brain, as in muscle. the purine nucleotide cycle might contribute to the regeneration of ATP under conditions which provokc a loss of high energy phosphatcs. This would also occur mainly by production of Krebs cycle intermediates.

5. MOLECULAR DEFECTS OF THE PURINE NUCLEOTIDE CYCLE In man, defects of the purine nucleotide cycle have been identified at the level of A M P deaminase and of adenylosuccinate lyase. A profound deficiency of muscle AMP deaminase (frequently referred to as myoadenylate deaminase in the clinical literature) was first described by Fishbein et al. (1978) in patients with muscular weakness or cramping after exercise. A regulatory mutation of liver AMP deaminase has been proposed as a cause of primary gout with overproduction of uric acid (Hers and Van den Berghe, 1979). Ogasawara et al. (1984b) discovered a complete, although totally asymptomatic deficiency oferythrocyte AMP deaminase in the Japanese population. Adenylosuccinate lyase deficiency was identified by Jaeken and Van den Berghe (1984) in three children with severe psychomotor retardation and autistic features, In this section the clinical, biochemical and pathophysiological aspects, genetics and treatment of these enzyme defects will be reviewed. 5.1. MUSCLEAMP DEAMINASEDEFICIENCY Numerous clinical reports and pathophysiologic studies have been devoted to this disorder (reviewed by Sabina et al., 1989b). Recently, it has also become the subject of extensive genetic investigations. 5.1. I. Clinical picture a n d diagnosis

Muscle AMP deaminase deficiency was initially described in five young adult males with muscular weakness, rapid fatigue, and cramps or myalgias following moderate to vigorous exercise (Fishbein et al., 1978). These symptoms were sometimes, but not always, accompanied by an increase in serum creatine kinase and minor electromyographic abnormalities. Muscular wasting and histologic abnormalities were absent. Later on however, wide variability was recorded with respect to both age of onset (1.5-70 years) and clinical symptoms of A M P deaminase deficiency. In addition, measurements of AMP deaminase activity in several large series of muscle biopsies, performed for diagnostic evaluation, have revealed that the enzyme activity is absent in about 2% of all specimens (Shumate et al., 1979; Kelemen et al., 1982; Mercelis et al., 1987). In these series, on average less than half of the AMP deaminasedeficient patients presented with the clinical picture described by Fishbein et al. (1978). Diagnoses in the other patients with the enzyme deficiency included, amongst others, amyotrophie lateral sclerosis, fascioscapulohumeral myopathy, Kugelberg-Welander syndrome, polyneuropathies and Werdnig-Hoffman

disease. It was therelore proposed that muscle AMP deaminase deficiency can be either a primary genetic defect, or acquired secondary to another neuromuscular disease (Fishbein, 1985). However, the finding of myoadenylate dcaminase deficicncy in asymptomatic thmily members of individuals with the defect (Sinkeler et al., 1988), combined with its frequency in diagnostic muscle biopsies and the absence of correlation with exercise intolerance (Mercelis et al., 1987) have led to two alternative hypotheses: (I) the deficiency is a common abnormality which has no clinical significance: (2) the deficiency becomes symptomatic only when an additional, hitherto unrecognized, inherited or acquired muscle abnormality is present or develops. Recent molecular biology studies (see Section 5.1.3) are in keeping with these hypotheses. Diagnosis of myoadenylate deaminase deficiency can be made in various ways. The lbrearm cxercise test, involving sponge-squeezing with a sphygmomanometer inflated to mean arterial pressure (Fishbein et al., 1978) is a sensitive screening procedure. In normal subjects, it provokes marked increases in venous plasma lactate and NH~. In myoadenylate deaminase deficiency, lactate increases normally but the elevation of NH~ is absent. Final diagnosis of the defect is established by histochemical (Fishbein et al., 1980) or biochemical assay of AMP deaminase in a muscle biopsy. 5.1.2. Biochemical a n d pathophysiological aspects

Measurements of myoadenylate deaminase activity in patients with the primary deficiency have shown activities below 2% of normal (Fishbein, 1985). Moreover, little or no immunoprecipitable enzyme was found and activities of other muscle enzymes, namely creatine kinase and adenylate kinase, were normal. In patients with secondary myoadenylate deaminase deficiency, its activity was between 2 and 15% of normal, as a rule appreciable immunoreactivity was present, and the activities of creatine kinase and adenylate kinase were usually below normal. A recent study of additional myoadenylate deaminasedeficient patients (Sabina et al., 1992) confirms these findings. The very limited capacity of myoadenylate deaminase-deficient muscle to form IMP, resulting in an elevation of AMP when the intensity of exercise is such that ATP decreases, has been documented (Sabina et al., 1980, 1984). In addition, the accumulation of AMP results in a net loss of adenine nucleotides by dephosphorylation of AMP into adenosine, followed by conversion into inosine and hypoxanthine. The extraction of energy from the adenylate pool is thus markedly diminished in AMP deaminase-deficient muscle, as also evidenced by the observation that the decrease in muscle creatine phosphate plus ATP per unit of work was five-fold greater in affected patients than in a control group (Sabina et al., 1984). These observations show impairment of the mechanisms whereby the purine nucleotide cycle has been proposed to regenerate ATP, discussed in Section 4.1. Paradoxically however, that A M P deaminase deficiency and consequent impairment of these ATP-regenerating mechanisms

PURINE NUCLEOTIOECYCLE

can be found in clinically asymptomatic individuals, indicates that they are not essential for normal muscle function. That plasma lactate increases to the same level as in control subjects in exercising myoadenylate deaminase-deficient patients shows that their production of energy by the glycolytic pathway proceeds normally. It also indicates that the release of NH 3 accompanying intense exercise does not play a role in the stimulation of muscle glycolysis under this condition. 5.1.3. Genetics Primary myoadenylate deaminase deficiency is most likely inherited as an autosomal recessive trait, as evidenced by the occurrence of the defect in families and in both sexes, and by the report of reduced activities of the enzyme in presumed heterozygotes (Kelemen et aL, 1982; Sinkeler et al., 1988). Families with pseudodominant inheritance have also been identified. This has been tentatively explained by the high frequency of heterozygotes in the population, which would also underly the finding of myoadenylate deaminase deficiency in about 2% of diagnostic muscle biopsies. The latter frequency, as discussed by Fishbein (1985), indicates a carrier incidence of about 20% according to the Hardy-Weinberg law. Recently, Gross et al. (1991) have reported that 12 unrelated Caucasian patients with primary myoadenylate deficiency were homozygous for two point mutations, located in exon 2 and 3, respectively, o f A M P D I cDNA. The mutation in exon 2 is a C--*T substitution located at nucleotide 34 which results in a non-sense mutation. That in exon 3 is also a C--,T substitution, which results in an exchange of Pro48--,Leu48. Expression experiments have, however, shown that this second mutation does not affect AMP deaminase activity. In accordance with findings in many other point mutations, Northern blot analyses in patients with primary myoadenylate deaminase deficiency have shown normal amounts of apparently normal-sized AMPD ! transcript (Sabina et al., 1992). Preliminary screening of 10 randomly selected healthy Caucasians has revealed that 2 of them were heterozygotes for both AMPD 1 mutations (Gross et al., 1991). This high, possibly 20% frequency of AMPD 1 heterozygocity in healthy individuals, would result in an approximately 2% incidence of AMP deaminase deficiency in the general population. That this figure accords with the finding that about 2% of diagnostic muscle biopsies are myoadenylate deaminase-deficient, reinforces the conclusion that there is no simple correlation of myoadenylate deaminase deficiency with exercise intolerance. 5.1.4. T r e a t m e n t

Administration of ribose (2 to 60 g per day orally in divided doses) has been reported very beneficial in some patients with myoadenylate deaminase deficiency (Patten, 1982; Z611ner et al., 1986) but ineffective in others (Lecky, 1983). Ribose may act as a glycolytic substrate and/or as a precursor of phosphoribosyl pyrophosphate, thereby stimulating de novo synthesis of purines (Wagner et al., 1991).

555

Patients with primary AMP deaminase deficiency have also been advised to exercise with caution to prevent rhabdomyolysis. 5.2. ERYTHROCYTEAMP DEAMINASEDEFICIENCY Systematic measurements of AMP deaminase in erythrocytes of blood donors led Ogasawara et al. (1984b) to identify virtual absence of its activity in four clinically and hematologically completely normal Japanese subjects. Family studies showed that the parents of the probands have a partial deficiency of erythrocyte AM P deaminase, indicating autosomal recessive inheritance. Moreover, individuals with approximately 50% of the control activity were found with a frequency of about !/30 in Japan and also in Korea and Taiwan (Ogasawara et al., 1987). Activities of AMP deaminase in mononuclear cells and in platelets are normal, confirming that the latter cell types contain a different isoform of the enzyme (Ogasawara et aL, 1984a). Activities of the other enzymes of purine catabolism and salvage, and of adenylate kinase are also normal. In the AMP deaminase-deficient erythrocytes concentrations of ATP are about 50% higher than normal, and remain higher for a longer time interval on storage. This can be explained by impairment of the degradation of AMP by way of AMP-deaminase, the rate-limiting enzyme of purine catabolism in these cells (Bontemps et al., 1986). Serum uric acid levels in the individuals with the enzyme defect tend to be slightly lower than in controls, probably as a result of slower purine catabolism in their red blood cells. 5.3. REGULATORY MUTATIONS OF LIVER A M P DEAMINASE

Among the human subjects with hyperuricemia and gout, the vast majority underexcrete uric acid owing to still unresolved renal defects, but about 10% overproduce uric acid (reviewed by Palella and Fox, 1989). In a very small minority of these overproducers, inborn errors of purine metabolism, namely hyperactivity of phosphoribosylpyrophosphate synthetase or partial or complete deficiency of hypoxanthine-guanine phosphoribosyltransferase are found. Both defects result in an increased de novo synthesis of purines. However, in most of the patients who overproduce uric acid, the mechanism of this overproduction is unknown. It has therefore been proposed (Hers and Van den Berghe, 1979) that regulatory mutations of liver AMP deaminase could be the underlying defect in a number of subjects with familial hyperuricemia due to overproduction of uric acid. These mutations would render the enzyme less sensitive to its physiological inhibitors, Pi and/or GTP. Owing to the 95% physiological inhibition of hepatic AMP deaminase by the levels of Pi and GTP prevailing in the liver cell under physiological conditions (Van den Berghe et al., 1977a), even a limited release of the inhibition could significantly increase the production of uric acid. Decreased sensitivity of AMP deaminase to its physiological inhibitors would also offer an explanation for the apparently dominant transmission of hyperuricemia

556

(J. VAN DEN BER(IHE et al.

in a number of families. Indeed, an excess of AMP deaminase activity would also be recorded when only 50% of the total enzyme protein is affected. Verification of the hypothesis requires complex kinetic analysis of hepatic AMP deaminase. These have hitherto only been performed in the autopsy liver of a single patient with florid gout, in whom a decreased sensitivity of the enzyme to GTP could be demonstrated (Van den Bcrghe and Hers, 1980). 5.4. ADENYLOSUCCINATELYASE DEFICIENCY This defect has been diagnosed up to now in 10 patients (Jaeken and Van den Berghe, 1984; Jaeken et al., 1988, 1992). 5.4.1. Clinical picture a n d diagnosis Two main subtypes of adenylosuccinate lyase deficiency can be distinguished: the first one, identified in 8 children and referred to as type I. is characterized by very severe psychomotor retardation. In addition, most of these children display autistic features such as failure to make eye-to-eye contact, repetitive movements and manipulations of objects, and occurrence of temper tantrums upon interference with repetitive behavior. Five of the patients also have epilepsy. Moreover, a brother and sister show marked weight and height growth retardation associated with muscular wasting. In type II, diagnosed in one girl, mental retardation is slight (Jaeken et al., 1988). In a third, recently identified variant, neurological involvement is intermediate (Jaekcn et al., 1992). Adenylosuccinate lyase deficiency results in thc accumulation in cerebrospinal fluid, plasma and urine of two normally undetectable compounds, SAICAriboside and succinyladenosine. These succinylpurines are the products of the dephosphorylation, by cytosolic 5'-nucleotidase (Van den Berghe and Jaeken, 1986), of the two substrates of the enzyme. In the profoundly retarded, type 1 patients, concentrations of both succinylpurines are 100--200,uM in cerebrospinal fluid, 5-10t~M in plasma and up to millimolar in urine, with succinyladenosine/ SAlCAriboside ratios between 1 and 2. In the markedly less retarded type II patient, concentrations of SAICAriboside are comparable to those in the type I patients. In contrast, succinyladenosine is markedly higher, resulting in succinyladenosine/ SAICAriboside ratios between 4 and 5 (Jaeken et al., 1988). The pronounced clinical heterogeneity of adenylosuccinate lyase deficiency justifies systematic screening for the enzyme defect in patients with unexplained mental retardation or neurological disease. For this purpose, a modified Bratton-Marshall test (Laikind et al., 1986a) performed on urine, is most practical. Final diagnosis is established by identification of the succinylpurines by high pressure liquid chromatography (Jaeken and Van den Berghe, 1984) and measurement of liver adenylosuccinate lyase activity. This should as a rule be performed on fresh biopsy specimens, owing to the sensitivity of the enzyme to freezing and thawing (Brand and Lowenstein, 1978; Van den Bergh et al., 1991a).

5.4.2. B w c h e m i c a l a n d pathophysiological aspects Measurements of the activity of adenylosuccinate lyase with adenylosuccinate in a series of patient tissues (Jaeken and Van den Berghe, 1984; Van den Berghe and Jaeken, 1986; Jaeken et al., 1988) have shown that the enzyme deficiency is not generalized. Adenylosuccinate lyase activity is reduced to 0-25% of normal in liver and kidney, and to approximately 40% in peripheral blood lymphocytes. It is normal in erythrocytes and granulocytes. A partial deficiency is also found in lymphoblasts generated l¥om the patients' lymphocytes (Barshop et al., 1989). The partial deficiencies of adcnylosuccinate lyase are probably explained by a decreased stability of the enzyme protein (Laikind et al., 1986b). The activity of adenylosuccinate lyase is also decreased in muscle of the patients with growth retardation and muscular wasting. Information concerning the activity of adenylosuccinate lyase in brain tissue of affected patients is lacking. Nevertheless, the 10- to 20-fold higher concentration of the succinylpurines in the cerebrospinal fluid of the patients as compared to plasma, provides a strong indication that the enzyme is also deficient in brain. The enzyme studies indicate the existence of isoforms of adenylosuccinate lyase in man, and agree with the clinical heterogeneity of the defect. Recent studies have shown that in the liver of the profoundly retarded, type I patients, the activities of adenylosuccinate lyase with its two substrates are lost in parallel (Van den Bergh et al., 1991a). This parallel loss is also found in cultured fibroblasts of type I patients: activities with adenylosuccinate and with SAICAR are both decreased to about 30% of the respective control values. In the type II patient, the activity with SAICAR was also approximately 30% of the control but, in marked contrast with the type I patients, that with adenylosuccinate was reduced to 3% of normal (Van den Bergh et al., 1991b). This differential reduction, if also present in other tissues, provides an explanation for the higher succinyladenosine/SAICAriboside ratio in the body fluids of the type 11, as compared to the type I patients. Two main hypotheses can be put forward to explain the pathophysiology of adenylosuccinate lyase deficiency. (I) The symptoms could result from deficiencies of the nucleotides which are normally formed distally from the enzyme defect. However, measurements in adenylosuccinate lyase-deficient freezeclamped tissues (Van den Berghe and Jaeken, 1986) and ~P magnetic resonance spectroscopy of the brain (Dorland et al., 1986) indicate that the defect does not result in decreased concentrations of purine nucleotides. Evidently, the enzyme deficiency might be compensated by recovery -by the enzymes of the salvage pathway--of purine bases supplied by the tissues in which adenylosuccinate lyasc activity is normal. Moreover, even low residual adenylosuccinate lyase activities may still permit purine nucleotide synthesis. In accordance with this hypothesis, intact fibroblasts of the type II patient were found to incorporate hypoxanthine via IMP and adenylosuccihate into adenine nucleotides, although at a slower rate than control cells (Van den Bergh et al., 1991b). As said in the previous paragraph, the residual

PURINE NUCLI~OT1DECYCLE

activity of adenylosuccinate iyase, measured with adenylosuccinate in fibroblast lysates of this patient, was only 3% of normal. Nevertheless, a deficiency of purine nucleotides could occur in cell types with low activities of the purine salvage enzymes. (2) The symptoms could be caused by accumulation of the substrates of adenylosuccinate lyase and/or of their dephosphorylated derivatives. Efficient dephosphorylation by cytoplasmic 5'-nucleotidase(s) probably explains why SAICAR and adenylosuccinate remain undetectable in adenylosuccinate lyase-deficient liver and muscle, although a slight accumulation of adenylosuccinate was measured in the kidney of two type I patients (Van den Berghe and Jaeken, 1986), and in the fibroblasts of the type II patient (Van den Bergh et al., 1991b). Nevertheless, deleterious effects could be caused by intracellular accumulations remaining below the level of detection of the methods used. From the observation of a strikingly less severe psychomotor retardation in the single patient with high succinyladenosine/SAICAriboside ratios, it is tempting to conclude that SAICAriboside is the offending compound and that succinyladenosine could protect against its toxic effects. Owing to the resemblance of both succinylpurines with adenosine, the possibility was explored that they might interfere with cerebral adenosine receptors, and thereby with the numerous physiologic functions of adenosine in brain (reviewed by Dunwiddie, 1985; Phillis, 1989). Studies with crude membrane fractions of rat cerebral cortex, however, failed to show interference of SAICAriboside and succinyladenosine with the binding of adenosine (Vincent and Van den Berghe, 1989). Measurements of the cerebral uptake of 6-tgfluoro-2deoxyglucose by positron emission tomography have shown that it is markedly reduced in the cortical areas of adenylosuccinate lyase-deficient patients (De Voider et al., 1988) suggesting interference of the succinylpurines with glucose metabolism. However, neither SAICAriboside nor succinyladenosine influenced glucose metabolism in isolated rat hepatocytes (M. F. Vincent and G. Van den Berghe, unpublished). 5.4.3. Genetics The occurrence of adenylosuccinate lyase deficiency in more than one child in two families, in patients of both sexes, and in two consanguineous marriages (Jaeken and Van den Berghe, 1984; Jaeken et al., 1988) indicates that the enzyme defect is transmitted as an autosomal recessive trait. Recent studies (Stone et al., 1991) have demonstrated a single T--*C substitution, predicting an exchange of Ser413--.Pro413 in two affected siblings. This substitution also creates a new Hph I restriction site which is diagnostic for the mutation. The marked variability of the clinical picture in adenylosuccinate lyase deficiency suggests that different mutations will most likely be found in the other affected families. 5.4.4. T r e a t m e n t With the aim of correcting hypothetically decreased concentrations of adenine nucleotides in adenylosuccinate lyase-deficient tissues, some

557

patients have been treated for several months with oral adenine (10 mg/kg per day), together with allopurinol (5-10 mg/kg per day) to inhibit its conversion into insoluble 2,8-OH-adenine. This treatment did not bring clinical or biochemical improvement, with the exception of weight gain and some acceleration of growth (Jaeken et al., 1988).

6. SUMMARY AND CONCLUSIONS Three enzymes of purine metabolism, adenylosuccinate synthetase, adenylosuccinate lyase and AMP deaminase, have been proposed to form a functional unit, termed the purine nucleotide cycle. This cycle converts AMP into IMP and reconverts IMP into AMP via adenylosuccinate, thereby producing NH3 and forming fumarate from aspartate. In muscle, the purine nucleotide cycle has been shown to function during intense exercise; the metabolic flux through the cycle has been proposed to play a role in the regeneration of ATP by pulling the adenylate kinase reaction in the direction of formation of ATP, and by providing Krebs cycle intermediates. In kidney, the purine nucleotide cycle was shown to account for the release of NH3 under the normal acid-base status, but not under acidotic conditions. In brain, the purine nucleotide cycle might function under conditions that induce a loss of ATP, and thereby contribute to its recovery. There is no evidence that the purine nucleotide cycle operates in liver. Deficiency of muscle AMP deaminase is an apparently frequent disorder, which might affect approximately 2% of the general population. The observation that it can be found in clinically asymptomatic individuals suggests, paradoxically, that the ATP-regenerating function which has been attributed to the purine nucleotide cycle is not essential for muscle function. Further work should be aimed at identifying the conditions under which AMP dcaminase deficiency becomes symptomatic. Adenylosuccinate lyase deficiency provokes psychomotor retardation, often accompanied by autistic features. Its clinical heterogeneity justifies systematic screening in patients with unexplained mental deficiency. Additional studies are required to determine the mechanisms whereby this enzyme defect results in psychomotor retardation. Acknowledgements--Work in the authors' laboratory was supported by the Fund for Medical Scientific Research (Belgium) and by the Belgian State--Prime Minister's Office for Science Policy Programming. G. Van den Bergh¢ is Director of Research of the Belgian National Fund for Scientific Research.

REFERENCES

ABRAMS,R. and BENTLEY,M. (1955) Transformation of inosinic acid to adenylic and guanylic acids in a soluble system. J. Am. Chem. Soc. 77, 4179--4180. AGARWAL,R. P. and PARKS,R. E. JR. (1977) Potent inhibition of muscle Y-AMP deaminase by the nucleoside antibiotics coformycin and deoxycoformycin. Biochem. Pharmac. 26, 663-666.

558

G. VAN DEN BERGHE et al.

AIMI, J., BADYLAK,J., WILLIAMS,J., CH~,N, Z., ZALKIN, H. and DIXON, J. E. (1990) Cloning of a eDNA encoding adenylosuccinate lyase by functional complementation in Escherichia coli. J. biol. Chem. 265, 9011-9014. ARAGON, J.J. and LOWENSTEIN, J. M. (1980) The purine nucleotide cycle. Comparison of the levels of citric acid cycle intermediates with the operation of the purine nucleotide cycle in rat skeletal muscle during exercise and recovery from exercise. Eur. J. Biochem. ll0, 371-377. ARCH, J. R. S. and NEWSHOLME, E. A. (1978) Activities and some properties of 5'-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine. Biochem. J. 174, 965-977. ASHBY, B. and FRIEDEN, C. (1978) Adenylate deaminase. Kinetic and binding studies on the rabbit muscle enzyme. J. biol. Chem. 253, 8728-8735. BARNES, L. B. and BISHOP, S. H. (1975) Adenylosuccinate lyase from human erythrocytes, bit. J. Biochem. 6, 497-503. BARSHOP, B. A. and FRIEDE,',:, C. (1984) Analysis of the interaction of rabbit skeletal muscle adenylate deaminase with myosin subfragments. A kinetically regulated system. J. biol. Chem. 259, 60-66. BAR,SHOP, B. A., ALBER'rS, A. S. and GRUBER. H. E. (1989) Kinetic studies of mutant human adenylosuccinase. Biochim. biophys. Acta 999, 19-23. BAUGHER, B. W., MONTONARO, L., WELCH, M. M. and RUDOLPH, F. B. (1980) Changes in isozymes of adenylosuccinate synthetase. Biochem. biophys. Res. Commun. 94, 123 129. BAUSCH-JURKEN, M. T., MAHNKI~-ZIZEI.MAN. D. K. and SABIr',;A,R. L. ( 1991 ) Characterization of the human AMP deaminase multigene family. Int. J. Purine Pvrimidine Res. 2, (suppl. I) 33. BOGUSKY, R. T., LOWENSTEIN,L. M. and LOWENSTEIN,J. M. (1976) The purine nucleotide cycle. A pathway for ammonia production in the rat kidney. J. olin. Invest. 58, 326--335. BOGUSKY,R. T., STEELE,K. A. and LOWENSTEIN,L. M. ( 1981 ) The purine nucleotide cycle in the regulation of ammoniagenesis during induction and cessation ofchronic acidosis in the rat kidney. Biochem. J. 196, 323-326. BOGUSKY, R. T., LOWENSTEIN. L. M. and AOKI, T. T. (1983) The relationship between glutamate deamination and gluconeogenesis in kidney. Biochem. J. 210, 695-698. BONTEMPS, F. and VAN DEN BERGHE,G. (1989) Mechanism of adenosine triphosphate catabolism induced by deoxyadenosine and by nucleoside analogues in adenosine deaminase-inhibited human erythrocytes. Cancer Res. 49, 4983-4989. BONTEMPS, F., VAN DEN BERGHE,G. and HERS, I'1. G. (1983) Evidence for a substrate cycle between AMP and adenosine in isolated hepatocytes. Proc. Dam. Acad. Sci. U.S.A. 80, 2829--2833. BONTEMPS, F., VAN DEN BERGHE,G. and HERS, H. G. (1986) Pathways of adenine nucleotide catabolism in erythrocytes. J. clin. Invest. 77, 824-830. BONTEMPS, F., VAN DEN BERGHE,G. and HERS, H. G. (1988) 5'-Nucleotidase activities in human erythrocytes. Identification ofa purine 5"-nucleotidase stimulated by ATP and glycerate 2,3-bisphosphate. Biochem. J. 250, 687-696. BRAND, L. M. and LOWENS'rEZN,J. M. (1978a) Effect of diet on adenylosuccinase activity in various organs of rat and chicken. J. biol. Chem. 253, 6872-6878. BRAND, L. M. and LOWENSTEIN,J. M. (1978b) Inhibition of adenylosuccinase by adenylophosphonopropionate and related compounds. Biochemistry 17, 1365--1370. CARTER, C. E. and COHEN, L. H. (1955) Enzymatic synthesis of adenylosuccinic acid. J. Am. Chem. Soc. 77, 499-500. C.~EY, P. J. and LOWENSTEIN,J. M. (1987a) Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects

of anions and fluoro analogues of the substrate. Biochem J. 246, 263-269. CASEY, P. J. and LOWENSTEIN.J. M. (1987b) Inhibition of adenylosuccinate lyase by L-alanosyl-5-aminoimidazole4-carboxylic acid ribonucleotide (alanosyl-AICOR). BiDchem. Pharmac. 36, 705-709. CASEY, P. J., ABELES, R. H. and LOWENSTEIN,J. M. (1986) Metabolism of threo-B-fluoroaspartate by H4 cells. Inhibition of adenylosuccinate lyase by fluoro analogs of its substrates. J. biol. Chem. 261, 13637--13642. CHANG, Z., NYGAARD, P., CHINAULT, A. C. and KELLEMS, R. E. ( 1991 ) Deduced amino acid sequence of Eseherichia co/i adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function. Biochemistry 30, 2273-2280. CLARK, S. W. and RUDOLPH, F. B. (1976) Regulation of purine metabolism. Adenylosuccinate synthetase from Novikoff ascites tumor cells. Biochim. biophys. Acta 437, 87-93. DE MOLDER,A. G.. JAEKEN.J., VAN DEN BERGHE,G., BOL, A., MICHEL, C., COGNEAU, M. and GOFFINET, A. M. (1988) Regional brain glucose utilization in adenylosuccinasedeficient patients measured by positron emission tomography. Pediatr. Res. 24, 238-242. DORLAND, L., VAN SPRANG, F. J., VAN ECHTELD, C. J. A., DURAN, M., WADMAN, S. K., DEN HOLLANDER,J. A. and LUYTEN, P. R. (1986) In vivo magnetic resonance spectroscopy and imaging of patients with adenylosuccinase deficiency. In: Annual Meeting o f the SSIEM, Abstract Book, p. 150. Amersfoort: The Netherlands. DUDLEY, G. A. and TERJt'NG, R. L. (1985) Influence of acidosis on AMP deaminase activity in contracting fasttwitch muscle. Am. J. Physiol. 248, C43 ~C50. DL'NWtDDIE, T. V. (1985) The physiological role of adenosine in the central nervous system. Int. Rev. Neurobiol. 27, 63 139. EMBDEN, G., RIEBI~LING,C. and SELTER, G. E. (1928) Ueber die Bedeutung der Adenyls/iure fuer die Muskelfunktion 2: Die Desaminierung der Adenylsaiire dutch Muskelbrei und die Ammoniakbildung bei der Muskelkontraktion. Hoppe-Se.),ler's Zeitschr. Physiol. Chemie 179, 149-160. FISHBEZN,W. N. (1985) Myoadenylate deaminase deficiency: inherited and acquired forms. Biochem. Med. 33, 158-169. FISHBEIN, W. N., ARMBRUSTMACHER,V. W. and GRIFFIN,J. L. (1978) Myoadenylate deaminase deficiency: a new disease of muscle. Science 200, 545 548. FISHBEIN, W. N., GRI~FtN, J. L. and ARMBRUSTMACHER.V. W. (1980) Stain for skeletal muscle adenylate deaminase. Arch. Path. Lab. Med. 104, 462 466. FLANAGAN,W. N., HOLMES,E. W., SABINA,R. L. and SwAts. J. L. (1986) Importance of purine nucleotide cycle to energy production in skeletal muscle. Am. J.Physiol. 251, C795-C802 F'OLBERGROVA, J., PASSONNEAU, J. V., [..()WRY, O. H. and SCHULTZ, D. W. (1969) Glycogen, ammonia and related metabolites in the brain during seizures evoked by sulfoximine. J. Neurochem. 16, 191 203. FRIEDEN, C., GILBERT, H. R., MILLER, W. H. and MILLER, R. L. (1979) Adenylate deaminase: potent inhibition by 2'-deoxycoformycin 5"-phosphate. Biochem. biophys. Res. Commun. 91, 278-283. GOODMAN, M. N. and IX)WENS'rEIN,J. M. (1977) The purine nucleotide cycle. Studies of ammonia production by skeletal muscle in situ and in perfused preparations. J. biol. Chem. 252, 5054-5060. GROSS, M., MORISAKI, H.. MORISAKI,T. and HOLMES, E. W. (1991) A common point mutation is responsible for AMP deaminase deficiency. Int. J. Purine Pyrimidine Res. 2, (Suppl. l): 50. GUICHERIT, O. M., RUDOLPH, F. B., KELLEMS, R. E. and COOPER, B. F. (1991) Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetasc. J. biol. Chem. 266, 22582--22587.

PURINE NUCLEOTIDECYCLE HERS, H. G. and VAN DEN BERGHE,G. (1979) Enzyme defect in primary gout. Lancet 1, 585-586. IKEGAMI,T., NAP3UMEDA,Y. and WEBER, G. (1989) Isozyme shift of adenylosuccinate synthase in rat and human neoplasms. Adv. exp. Med. Biol. 253A, 417--421. ITO, K., YAMAMOTO,H. and MIZUGAKI, M. (1988) Purification and general properties of AMP deaminasc from sheep brain. J. Biochem. (Tokyo) 103, 259-262. JAEKEN, J. and VAN DEN BERGrrE, G. (1984) An infantile autistic syndrome characterised by the presence of succinylpurines in body fluids. Lancet 2, 1058-1061. JAEKEN, J., WADMAN, S. K., DURAN, M., VAN SPRANG, F. J., BEEMER,F. A., HOLL, R. A., THEUNISSEN,P. M., DE COCK, P., VAN DEN BERGH, F., VINCENT, M. F. and VAN DEN BERGHE. G. (1988) Adenylosuccinase deficiency: an inborn error of purine nucleotide synthesis. Eur. J. Pediatr. 148, 126-131. JAEKEN, J., VAN DEN BERGH, F., VINCENT, M. F., CASAER,P. and Van DEN BERGHE, G. (1992) Adenylosuccinase deficiency: a newly recognized variant. J. inher, metab. Dis., in press. KALETHA, K. and NOWAK, G. (1988) Developmental forms of human skeletal-muscle AMP deaminase. The kinetic and regulatory properties of the enzyme. Bioehem. J. 2,19, 255-261. KALETHA, K., SPYCHALA,J. and NOWAK, G. (1987) Developmental forms of human skeletal muscle AMP deaminase. Experientia 43, 440-M43. KELEMEN, J., RICE, D. R., BRADLEY,W. G., MUNSAT, T. L., DIMAURO, S. and HOGAN, E. L. (1982) Familial myoadenylate deaminase deficiency and exertional myalgia. Neurology 32, 857-863. KREBS, H. A., HEMS, P., LUND, P., HALLIDAY,D. and READ, W. W. C. (1978) Sources of ammonia for mammalian urea synthesis. Biochem. J. 176, 733-737. LAI, L., HART, I. and PATTERSON,D. (1989) Human chromosome 1 corrects the defect in the CHO mutant (Ade-") deficient in a branch point enzyme in purine de novo biosynthesis. Cytogenet. Cell Genet. 51, 1028. LAIKIND, P. K., SEEGMILLER,J. E. and GRUBER,H. E. (1986a) Detection of 5'-phosphoribosyl-4-(N-succinylcarboxamide)-5-aminoimidazole in urine by use of the BrattonMarshall reaction: identification of patients deficient in adenylosuccinate lyase activity. Anal. Biochem. 156, 81-90.

LAIKIND, P. K., GRUBER, H. E., JANSEN, I., MILLER, U, HOFFER, M., SEEGMILLER,J. E., WILLIS,R. C., JAEKEN,J. and VAN DEN BERGHE,G. (1986b) Purine biosynthesis in chinese hamster cell mutants and human fibroblasts partially deficient in adenylosuccinate lyase. Adv. exp. Med. Biol. 195B, 363-369. LECKY, B. R. F. (1983) Failure of D-ribose in myoadenylate deaminase deficiency. Lancet 1, 193. LEE, Y.-P. and WANG, M.-H. (1968) Studies of the nature of the inhibitory action of inorganic phosphate, fluoride, and detergents on 5'-adenylic acid deaminase activity and on the activation by adenosine triphosphate. J. biol. Chem. 243, 2260-2265. LIAN, C.-Y. and HARKr,W.SS,D. R. (1974) The kinetic properties of adenylate deaminase from human erythrocytes. Biochim. biophys. Acta 341, 27-40. LOWENSTEIN, J. M. (1972) Ammonia production in muscle and other tissues: the purine nucleotide cycle. Physiol. Rev. 52, 382-414. L,OWENS'rEIN, J. M. and TORNHEIM, K. (1971) Ammonia production in muscle: the purine nucleotide cycle. Science 171, 397-400. LOWENSTEIN,J. M. and GOODMAN,M. N. (1978) The purine nucleotide cycle in skeletal muscle. Federation Proc. 37, 2308-2312. LowY, B. A., WILLIAMS, M. K. and LONDON, I. M. (1962) Enzymatic deficiencies of purine nucleotide synthesis in the human erythrocyte. J. biol. Chem. 237, 1622-1625.

559

MANFREDI, J. P. and HOLMES, E. W. (1984) Control of the purine nucleotide cycle in extracts of rat skeletal muscle: effect of energy state and concentrations of cycle intermediates. Arch. Biochem. Biophys. 233, 515-529. MARQUETANT, R., DESAI, N. M., SABINA,R. L. and HOLMES, E. W. (1987) Evidence for sequential expression of multiple AMP deaminase isoforms during skeletal muscle development. Proc. nam. Acad. Sei. U.S.A. 84, 2345-2349. MARQUETANT, R., SABINA, R. L. and HOLMES, E. W. (1989) Identificationof a noncatalytic domain in A M P dcaminase that influencesbinding to myosin. Biochemistry 28, 8744-8749. MATSUDA, Y., OGAWA, H., FUKUTOME,S., SHIRAKI, H. and NAKAGAWA,H. (1977) Adenylosuccinate synthetase in rat liver: the existence of two types and their regulatory roles. Biochem. biophys. Res. Commun. 78, 766-771. MATSUDA, Y., SHIRAKI, H., OGAWA,H. and NAKAGAWA,H. (1978) Change in content of adenylosuccinate synthetase isozymes during liver regeneration in rats. 8ioehim. biDphys. Acta 541, 135-138. McGIVAN, J. D. and CHAPPELL, J. B. (1975) On the metabolic function of glutamate dehydrogenase in rat liver. FEBS Left. 52, I-7. MERCELIS, R., MARTIN, J.-J., DE BARSY, T. and VAN DEN BERGHE, G. (1987) Myoadcnylate deaminase deficiency: absence of correlation with exercise intolerance in 452 muscle biopsies. J. Neurol. 234, 385-389. MEYER, R. A. and TERJUNG. R. L. (1979) Differences in ammonia and adenylate metabolism in contracting fast and slow muscles. Am. J. Physiol. 237, C I I I - - C l l H . MEYER, R. A.. DUDLEY, G. A. and TERJUNG, R. L. (1980) Ammonia and IMP in different skeletal muscle fibers after exercise in rats. J. appl. Physiol. 49, 1037-1041. MINED, I., CLARKE, P. R. H., SABINA, R. L. and HOLMES, E. W. (1990) A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPDI. Molec. cell. Biol. 10, 5271-5278. MOMMSEN,T. P. and HOCHACHKA, P. W. (1988) The purine nucleotide cycle as two temporally separated metabolic units: a study on trout muscle. Metabolism 37, 552-556. MORISAKI, T., SABINA, R. L. and HOLMES, E. W. (1990) Adenylate deaminase. A multigene family in humans and rats. J. biol. Chem. 265, 11482-11486. Moss, K. M. and McGIVAN, J. D. (1975) Characteristics of aspartate deamination by the purine nucleotide cycle in the cytosol fraction of rat liver. Biochem. J. 150, 275-283. NISSIM, I., YUDKOFF,M. and SEGAL,S. (1985) Metabolism of glutaminc and glutamate by rat renal tubules. Study with ~SN and gas chromatography-mass spectrometry. J. biol. Chem. 260, 13955-13967. NISSlM, I., YUDKOFE, M. and SEGAL, S. (1986) Effect of 5-amino-4-imidazolecarboxamide riboside on renal ammoniagenesis. Study with [~SN]aspartate. J. biol. Chem. 261, 6509-6514. OGASAWARA, N., GOTO, H. and WATANABE, T. (1975a) Isozymes of rat AMP deaminase. Biochim. biophys. Acta 403, 530-537. OOASAWARA, N., GOTO, H. and WATANABE, T. (1975b) lsozymes of rat brain AMP deaminase: developmental changes and characterizations of five forms. FEBS Lett. 58, 245-248. OGASAWARA, N., GOTO, H., YAMADA, Y. and WATANABE,T. (1978) Distribution of AMP-dcaminase isozymcs in rat tissues.Fur. J. Biochem. 87, 297-304. OGASAWARA, N., GOTO, H., YAMADA, Y., WATANABE, T. and ASANO, T. (1982) A M P deaminasc isozymes in human tissues. Biochim. biophys. Acta 714, 298-306. OGASAWARA,N., GOTO, H., YAMADA,Y. and WATANABE,T. (1984a) Distribution of A MP-deaminase isozymes in various human blood cells. Int. J. Biochem. 16, 269-273. OGASAWARA, N., GOTO, H., YAMADA, Y., NISHIGAKI, !., ITOH,T. and HASEGAWA,I. (1984b) Complete deficiency of

560

G. VAN DEN BERGHEet al.

AMP deaminase in human erythrocytes. Biochem. biDphys. Res. Commun. 122, 1344-1349. OGASAWARA, N., GOT(), H., YAMADA, Y., NISHIGAKI, 1., ITOH,T., HASEGAWA,I. and PARK,K. S. (1987) Deficiency of AMP deaminase in erythrocytes. Hum. Genet. 75, 15 18. OGAWA, H., SHmAKI, H. and NAKAGAWA,H. (1976) Study on the regulatory role of fructose-l,6-diphosphate in the formation of AMP in rat skeletal muscle. A mechanism for synchronization of glycolysis and the purine nucleotide cycle. Biochem. biophys. Res. Commun. 68, 524 528. PALELLA, T. D. and Fox, I. H. (1989) Hyperuricemia and gout. In: The Metabolic Basis o f Inherited Disease 6th ed., pp. 965-1006. Eds. C. R. SCRIVER,A L. BEAUI)ET,W. S. SLY and D. VALLE. McGraw-Hill, New York. PARNAS, J. K. and MOZOLOWSKI, W. (1927) Ueber den Ammoniakgehalt und die Ammoniakbildung im Muskel und deren Zusammenhang mit Funktion und Zustandsaenderung. Biochem. Zeitschr. 184, 399-441. PATTEN, B. M. (1982) Beneficial effect of D-ribose in patient with myoadenylate deaminase deficiency. Lancet I, 1071. PHILLIS,J. W. (1989) Adenosine in the control of the cerebral circulation. Cerebrovasc. Brain Metab. Rev. I, 26-54. POWELL, S. M., ZALKIN, H. and DIXON,J. E. (1991) Cloning of human cDNA for adenylosuccinate synthetase. Clin. Res. 39, 705A. RA'rNER, S. (1972) Argininosuccinases and adenylosucciBases. In: The Enzymes, 3rd ed.. Vol. 7. 167-197. ROGNSTAD,R. (1977) Sources of ammonia for urea synthesis in isolated rat liver cells. Biochim. biophys. Acta 496, 249--254. RONCA-TESTONI,S., RAGGI.A. and RONCA,G. (1970) Muscle AMP aminohydrolase. 111. A comparative study on the regulatory properties of skeletal muscle enzyme from various species. Biochim. biophys. Acta 198, 101-112. SABINA, R. L., SWAIN,J. L., PATTEN, B. M., ASHIZAWA,T., O'BRIEN, W. E. and HOLMES,E. W. (1980) Disruption of the purine nucleotide cycle. A potential explanation for muscle dysfunction in myoadenylate deamina~ deficiency. J. clin. Invest. 66, 1419-1423. SABINA,R. L., KERNSTINE,K. H., BOYD,R. L., HOLMES,E. W. and SWAIN,J. L. (1982) Metabolism of 5-amino-4-imidazolecarboxamide riboside in cardiac and skeletal muscle. Effects on purine nucleotide synthesis. J. biol. Chem. 25% 10178 10183. SABINA, R. L., SWAIN, J. L., OLANOW, C. W., BRADLEY, W. G., FISHBEIN,W. N., DIMAURO, S. and HOLMES,E. W. (1984) Myoadenylate deaminase deficiency. Functional and metabolic abnormalities associated with disruption of the purine nucleotide cycle. J. clin. Invest. 73, 720-730. SABINA, R. L., PATTERSON, D. and HOLMF,S, E. W. (1985) 5-Amino-4-imidazole-carboxamide riboside (Z-riboside) metabolism in eukaryotic cells. J. biol. Chem. 260. 6107~5114. SABINA, R. L., OGASAWARA,N. and HOLMES,E. W. (1989a) Expression of three stage-specific transcripts of AMP deaminase during myogenesis. Molec. cell. Biol. 9, 2244-2246. SABtNA, R. L., SWAIN, J. L. and HOLMES, E. W. (1989b) Myoadenylate deaminase deficiency. In: The Metabolic Basis of Inherited Disease 6th ed.. pp. 1077 1084. Eds. C. R. SCRIVER,A. L. BEAUDET,W. S. SLY and D. VALLE. McGraw-Hill: New York. SABINA, R. L., MORISAKI,T., CLARKE,P., EDDY, R., SHOWS, T. B., MORTON,C. C. and HOLMES,E. W. (1990) Characterization of the human and rat myoadenylate deaminasc genes. J. biol. Chem. 265, 9423-9433. SABINA, R. L., FISHBEIN,W. N., PEZESHKPOUR,G., CLARKE, P. R. H. and HOLMES,E. W. (1992) Molecular analysis of the myoadenylate deaminase deficiencies. Neurology, in press. SCHMIDT, G. (1928) Ueber fermentative Desaminierung im Muskel. Hoppe-Seyler's Zeitschr. Physiol. Chemic 179, 243-282.

SHULTZ, V. and LOWENSTEIN.J. M. (1976) Purine nucleotide cycle. Evidence for the occurrence of the cycle in brain. J. biol. Chem. 251,485 492. SCrlULTZ, V. and LOWENSTEIN, J. M. (1978) The purIne nucleotide cycle. Studies of ammonia production and interconversions of adenine and hypoxanthine nude()tides and nuc[eosides by rat brain in situ. J. biol. Chem. 253, 1938-1943. SETLOW. B. and LOWENSTEIN.J. M. (1967) Adenylate deaminase. Ii. Purification and some regulatory properties of the enzyme from calf brain. J. biol. Chem. 242, 607.~515. SETLOW, B. and LOWENSTEIN,J. M. (1968) Adenylate deaminase. IV. Nucleotide specificity of the enzyme from calf brain with special reference to guanosine triphosphate. J. biol. Chem. 243, 3409-3415. SHIGEt'RA, H. T. and GORDON, C. N. (1962) The mechanism of action of hadacidin. J. biol. Chem. 237, 1937-1940. SHIRAKI, H., MIYAMOTO,S., MATSUDA,Y., MOMOSE,E. and NAKAGAWA,H ( 1981 ) Possible correlation between binding of muscle type AMP deaminase to myofibrils and ammoniagenesis in rat skeletal muscle on electrical stimulation. Biochem. biophys. Res. Commun. 100, 1099- 1103. SHUMATE,J. B., KATNIK,R., RuIz. M., KAIZER,K., FRIEDEN. C., BROOKE,M. H. and CARROLL,J. E (1979) Myoadenylate deaminase deficiency. Muscle Nerve 2, 2i3 216. SINKELER, S. P. T., J(X)STEN, E. M. G., WEVER, R. A., OEI, T. L, JACOBS, A. E. M., VEERKAMP.J. H. and IIAMEL, B. C. J. (1988) Myoadenylate deaminase deficiency: a clinical, genetic and biochemical study in nine families. Muscle Nerve I 1, 312 317. SMILEr, K. L., JR and SUELIER, C. H. (1967) Univalent cations as allosteric activators of muscle adenosine 5"phosphate deaminasc. J. biol. Chem. 242, 1980-1981. SMITH, L. D., EMERSON,R. L. and MORRICAL,S. W. (1982) A comparison of hepatic adenylosuecinate lyase from rats fed either a chow diet or a ~misynthetic basal diet low in riboflavin. Int. J. Biochem. 14, 875-878. STAYTON, M. M., RUDOLPH, F. B. and FROMM,H. J. (1983) Regulation, genetics and properties of adenylosuccinate synthetase: a review. Curr. Top. cell. Regul. 22, 103-141. STONE. R. L., AIMI,J., ZALKIN,H., DIXON,J. E. and BARSHOP, B. A. (1991) A genetic defect in the adenylosuccinate lyase mRNA associated with infantile autism. FASEB J. 5, A 1510. TOMOZAWA, Y. and WOLFENt)EN, R. (1970) Binding of guanosine triphosphate and adenosine triphosphate by rabbit muscle adenosine monophosphate deaminase. Biochemistry 9, 3400--3404. TORNm~IM, K. and LOWENSTEIN,J. M. (1972) The purine nucleotide cycle. The production of ammonia from aspartate by extracts of rat skeletal muscle. J. biol. Chem. 247, 162--169. TORNHEIM, K. and IG)WENSTEIN, J. M. (1973) The purine nucleotide cycle. Ill. Oscillations in metabolite concentrations during the operation of the cycle in muscle extracts. J. biol. Chem. 248, 2670 2677. TORNHEIM, K., PANG, H. and COSTELLO, C. E. (1986) The purine nucleotide cycle and ammoniagenesis in rat kidney tubules. J. biol. Chem. 261, I0157-I0162. TRUONG, V. L., COLLINSON, A. R. and LOWENSTEIN,J. M. (1988) 5'-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities. Biochem. J. 253, 117-121. TULLSDN.P. C., WrlITL(XZK,D. M. and TERJUNG,R. L. (1990) Adenine nucleotide degradation in slow-twitch red muscle. Am. J. Physiol. 258, C258-C265. TYAGI, A. K. and COONEY,D. A. (1980) Identification of the antimetabolite of L-alanosine, L-alanosyl-5-amino-4imidazolecarboxylic acid ribonucleotide, in tumors and assessment of its inhibition of adenylosuccinate synthetase. Cancer Res. 40, 4390-4397. VAN DEN BERGH,F., VINCENT,M. F., JAEKEN)J. and VANDEN BERGHEG. (1991a) Radiochemical assay of adenylosucci-

PURIN'E NUCLEOTIDECYCLE nase: demonstration of parallel loss of activity toward both adenylosuccinate and succinylaminoimidazole carboxamide ribotide in liver of patients with the enzyme defect. Analyt. Biochem. 193, 287-291. VAN DEN BERGH,F., VINCENT,M. F., JAEKEN,J. and VAN DEN BERGHE, G. (1991b) Adenylosuccinase activity and succinylpurine production in fibroblasts of adenylosuccinasedeficient children. Adv. exp. Med. Biol. 309B, 277 -280. VAN DEN BERGHE, G. (1986) Fructose: metabolism and short-term effects on carbohydrate and purine metabolic pathways. Prog. Biochem. Pharmac. 21, 1-32. VAN DEN BERGrn~, G. and HERS, H. G. (1980) Abnormal AMP deaminase in primary gout. Lancet 2, 1090. VAN DEN BERGHE, G. and JAEKEN, J. (1986) Adenylosuccinase deficiency. Adv. exp. Med. Biol. 195A, 27-33. VAN DEN BERGHE, G., BRONFMAN, M., VANNESTE, R. and HERS, H. G. (1977a) The mechanism of adenosine triphosphate depletion in the liver after a load of fructose. A kinetic study of liver adenylate deaminase. Biochem. J. 162, 601-609. VAN DEN BERGHE, G., VAN POTTELSBERGHE, C. and HERS. H. G. (1977b) A kinetic study of the soluble 5'-nucleotidase of rat liver. Biochem. J. 162, 611-616. VAN DEN BERGHE,G., BONTEMPS,F. and HERS, H. G. (1980) Purine catabolism in isolated rat hepatocytes. Influence of coformycin. Biochem. J. 188, 913 -920. VAN DEN BERGHE, G., BONTEMPS, F. and VINCENT, M. F. (1988) Cytosolic purine 5'-nucleotidases of rat liver and human red blood cells: regulatory properties and role in AMP dephosphorylation. Adv. Enzyme Regul. 27, 297-311. VAN KEUREN, M. L., HART. I. M., KAO, F.-T., NEVE, R. L., BRUNS, G. A. P., KURN1T,D. M. and PATTERSON,D. (1987) A somatic cell hybrid with a single human chromosome 22 corrects the defect in the CHO mutant (Ade !) lacking adenylosuccinase activity. Cytogenet. Cell Genet. 44, 142- 147. VAN WAARDE,A. (1988) Operation of the purine nucleotide cycle in animal tissues. Biol. Ret'. 63, 259 298.

561

VINCENT, M. F. and VAN DEN BERGFIE0G. (1989) Influence of succinylpurines on the binding of adenosine to a particular fraction of rat cerebral cortex. Adv. exp. Med. Biol. 253B, 441-445. VINCENT,M. F., VAN DEN BERGITEG. and HERS, H. G. (1989) D-Xylulose-induced depletion of ATP and Pi in isolated rat hepatocytes. FASEB J. 3, 1855-1861. VINCENT, M. F., MARANGOS,P. J., GRUBER,H. E. and VAN DEN BERGHE,G. (1991) Inhibition by AICA riboside of gluconeogenesis in isolated rat hepatocytes. Diabetes 40 1259-1266. WAGNER, D. R., GRESSER, U., KAMILLI,l., GROSS, M. and ZOLLNER, N. (1991) Effects of oral ribose on muscle metabolism during bicycle ergometer in patients with myoadenylate deaminase deficiency. Int. J. Purine Pyrimidine res. 2, (suppl. I) 96. WHEELER, T. J. and LOWENSTEIN, J. M. (1979) Adenylate deaminase from rat muscle. Regulation by purine nucleotides and orthophosphate in the presence of 150 mM KCI. J. biol. Chem. 254, 8994-8999. YOSHINO, M., MURAKAMI,K. and TSUSmMA, K. (1978) The role of polyamines in the regulation of AMP deaminase isozymes. Biochim. biophys. Acta 542, 177-179. YOSHINO, M., MIYAJ1MA,E. and TSUSHIMA, K. (1979) Kinetics of the interactions of AMP deaminase with fatty acids. J. biol. Chem. 254, 1521 1525. ZIELKE, C. L. and SUELTER, C. H. (1971a) Purine, purine nucleoside and purine nucleotide aminohydrolases. In: The Enzymes 3rd ed., Vol. 4, 47-78. ZIELKE, C. L. and SUELTER,C. H. (1971b) Substrate specificity and aspects of deamination catalyzed by rabbit muscle 5'-adenylic acid aminohydrolase. J. biol. Chem. 246, 1313-1317. ZOLLNER, N., RE.ITER, S., GROSS, M., PONGRATZ, D., REIMERS, C. D., GERBITZ, K., PAETZKE, l., DEL'I-EL, T. and HUBNER, G. (1986) Myoadenylate deaminase deficiency: successful symptomatic therapy by high dose oral administration of ribose. Klin. Wochenschr. 64, 1281-1290.