Enzymological aspects of disorders in purine metabolism

31

Enzymological Aspects of Disorders in Purine Metabolism MATHIAS M. MULLER, MARTIN KRAUPP, and PETER CHIBA 2nd Department of Medicine, University of Vienna, Garnisongasse 13, A-1090 Vienna, Austria Department of Medical Chemistry, University of Vienna, Wahringerstral3e 10, A-1090 Vienna, Austria Congenital enzyme defects of purine synthesis de novo and the salvage pathway are responsible for excessive uric acid production and are often associated with hyperuricemia and gout. On the other hand, defects of enzymes essential for the purine nucleotide cycles are the biochemical basis of dysfunction of the immune system. The influence of several congenital enzyme deficiencies on the regulation of biosynthesis de novo, on the regulation of purine nucleotide concentrations, and on adenosine concentration, as well as the effect on purine transport through cell membranes are discussed. The determination of enzymes involved in purine metabolism in noncongenital diseases seems to be of diagnostic importance. As examples, enzyme activities in lymphocytes of leukemic patients, and the determination of serum guanase activity in patients with liver dysfunction are described.

he study of purine metabolism is a rapidly expandT ing field. The present communication will deal with enzymological aspects of congenital as well as noncongenital or acquired diseases. The consequences for the regulation of purine metabolism and for the transport of purines are described. For the clinical enzymologist these enzymatic peculiarities increasingly become of interest h'om the diagnostic point of view. 1. D e f e c t s in c o n g e n i t a l d i s e a s e s Some genetically determined enzyme defects (inborn errors of purine metabolism) are listed in Table 1. These enzyme defects are expressed in all somatic cells, except for xanthine oxidase ~XOX, EC 1.3.2.3), which is present in considerable amounts only in liver and small intestinal mucosa ( 1 ). It should, however, be mentioned that the determination under optimal conditions of residual activities of m u t a n t enzymes with lowered catalytic capacity does not give much information on the relation between enzymatic activity and the clinical picture. Recently a patient with full clinical expression of the Lesch-Nyhan syndrome ILNS) was described who showed 30(~ residual H G P R T activity in his fibroblast lysates (21. Usually in LNS fibroblasts residual activi-

ties of H G P R T are a few percent of normal 131. The m u t a n t enzyme of" this patient had an increased apparent KM for PRPP and decreased thermostability. In view of the intracellular PRPP levels, it was concluded that the activity of the m u t a n t enzyme under in uivo conditions must be virtually zero (2). Not only decreased enzyme activities, but also one example of increased activity of a m u t a n t enzyme has been reported: the superactivity of PRPP-S. The h'equency of occurrence of the mentioned inborn errors is not yet exactly known, but it amounts presumably to less than 1 : 100,000 live births for each. However, due to the devastating effects of these genetic diseases, heterozygote detection and prenatal diagnosis by means of enzymological analysis are indicated (4). A n u m b e r of enzyme defects expressed only in certain tissues are listed in Table 2. These cause hematological disturbances and muscular disorders. The abnormal (both increased and decreased) activities in erythrocytes of patients with certain forms of hemolytic anemia might reflect the presence of specific subfractions of erythrocytes that are characterized by these enzyme levels. The same holds for x-linked a g a m m a g l o b u linemia: the decreased ecto-5'PNT 15) might reflect the presence of a subfraction of lymphocytes characterized by low levels of 5'-PNT. The enzyme defects listed in Tables 1 and 2 might cause a disturbance of the regulatory mechanism of purine metabolism, or an impairment of purine transport through the cell m e m b r a n e . 1.1. DEFECTS AND REGULATIONOF PURINE METABOLISM The regulatory mechanisms which operate at different sites of purine metabolism involve control by substrate concentrations and feedback control (Figure 1). Besides, control of enzyme turnover as demonstrated in bacterial systems (6) might be a simple mechanism for controlling quantities of enzymes I7) and metabolic rates.

TABLE 1 Enzyme Defects of Purine Metabolism in Congenital Diseases Expressed In All Tissues

Enzyme A.

B.

Clinical Picture

Decreased enzyme activity: hypoxanthine guanine phosphoribosyltransferase (HGPRT) severe deficiency partial deficiency adenine phosphoribosyltransferase IAPRTI adenosine deaminase (ADA) purine nucleoside phosphorylase (PNP) xanthine oxidase (XOX)

Lesch-Nyhan syndrome gout kidney stones severe combined immunodeficiency dysfunction of T-lymphocytes xanthinuria

Increased enzyme activity phosphoribosylpyrophosphate synthetase (PRPP-S)

gout

32

MULLER, KRAUPP AND CHIBA RIBOSE-5-PHOSPHATE

PRPP-AMIDOTRANSFERASE]

PNOSMmOSXLAW dATP

ATP

GTP

,16TP RIIOSE" 5 - P

dADP~AOP iir.|i---

ill

PRPP ,

.-

GOP~aGDP

ADENINE

1

l iMP _

INOSINE HY~XAI(THINE

l

PRPP

GMP

upnv PRPP ~

I/

1[-5:''~J GUAHO~IHE

J

l t~

"PURINE NUCLEOTIDES MW

GOMIllE

iNACTIVE

XANTHm

1

T

2 7 0 OOO

|0I

2.8"DIND~I~ADENINE

ATp

DE UO¥O STNTNESIS

MWI~O00

SALVAGE PATNWAY

URICACIO

Figure 1 -- Pathways of purine metabolism. 1 . I . I . R e g u l a t i o n a , d biosynthesis de novo

The reaction catalyzed by phosphoribosylpyrophosphate-amidotransferase (PRPP-AT, E.C. 2.4.2.14) is considered the rate-limiting step of purine biosynthesis. The activity of PRPP-AT is modulated by the concentrations of phosphoribosylpyrophosphate (PRPP) and of purine nucleotides acting as allosteric inhibitors. The affinities of various PRPP-consuming enzymes establish a priority system: the KM-value for adenine phosphoribosyl transferase (APRT, E.C. 2.4.2.7) is the lowest, that of hypoxanthine guanine phosphoribosyltransferase (HGPRT, E.C. 2.4.2.8) intermediate, and that of PRPP-AT highest 18). Taking the intracellular PRPP concentration into consideration, none of the enzymes mentioned would be saturated. However, APRT and HGPRT will be favoured in competing for PRPP over PRPP-AT. Therefore, depletion of intracellular PRPP by the addition of preformed exogenous purines will diminish de novo synthesis (9), while an increase in PRPP because of a deficiency of a PRPP-consuming reaction (e.g. HGPRT deficiency in LNS) will be followed by an increased rate of de novo synthesis (10). Since the substrate-velocity curve for PRPP shows sigmoidal characteristics (11), changes of

PRPP concentration may greatly influence the formation of phosphoribosylamine. The second mechanism involved in the regulation of PRPP-AT activity is allosteric feedback inhibition by nucleotides. Both hydroxy- and adenine nucleotides occupy different allosteric sites on the enzyme surface, and inhibit the enzyme synergistically (Figure 2). Nucleotides convert, by aggregation, the two smaller subunits to the inactive large form (12). Since intracellular concentrations of purine nucleotides (adenine nucleotide concentration: 2.7 × 10 :' M (13)) are very close to their K,-values for PRPP-AT 114}, this might indicate that PRPP-AT is normally under considerable inhibition by nucleotides. This synergistic inhibition by purine nucleotides is completely overcome by high PRPP concentrations leading to disaggregation of the subunits (121. Phosphoribosylpyrophosphate synthetase (PRPP-S, E.C. 2.7.6.1.) might be another rate-limiting site in de novo purine synthesis (Figure 3). ADP, PRPP and 2,3-diphospho-glycerate (2,3-DPG)inhibit PRPP-S in a competitive m a n n e r with respect to MgATP and ribose5-phosphate respectively (15). Since the K,-value for PRPP is 10 times higher than its intracellular concentration, this inhibition seems unlikely to be of biological importance. In contrast, 2,3-DPG intracellular concentrations are in the range of its K,-value. Furthermore 2,3-DPG suppresses PRPP-S activity by disaggregating the enzyme to inactive small aggregates or monomer forms (16). ADP has a K,-value below its

TABLE 2 Enzyme Defects of Purine Metabolism in Congenital Diseases Expressed In Certain Cell Types

Clinical Picture

Affected Cells/Tissue erythrocytes erythrocytes muscle

purine-5'-nucleotidase (5'-PNT)

hemolytic anemia hemolytic anemia malignant hyperthermia after anaesthesia muscular weakness and cramping after exercise x-linked agammaglobulinemia

Increased Enzyme Activity adenosine deaminase (ADA)

hemolytic anemia

erythrocytes

A. Decreased Enzyme Activity adenosine triphosphatase (ATPase) adenylate kinase adenylate deaminase

B.

ACTIVE

Figure 2 - - Regulation of PRPP-amidotransferase activity.

1,°,

Enzyme

~IW 1 ~ 0 0 (

muscle lymphocytes

ENZYMOLOGICAL ASPECTS OF DISORDERS IN PUR1NE METABOLISM AOP

2,3-DPG

~

ADENOSINE-TRIPHOSPHATE~

"U:

RIBOSE'5"PHOSPNATE

AMP, . . . . . .

....

ADENYLO ,~ [~---:~ SUCCINATE ",,

33

liE l o l l SIITNiIll

XMP . . . . . .

IMP

~ GMP

Ku 4o0

INO

,I.I URIC ACID

PRPP

PYRIMIDINENUCLEOTIDES

PURINE o NUCLEOTIDES

I

competitive inhibition

I~

noncompetitive inhibition (feed-back control )

KM- VALUES:

prnol/I

SW~ZER,,j ~zl

Figure 3 -- Feedback control of phosphoribosylpyrophosphate synthetase. physiological concentration and changes the substrate velocity plot from hyperbolic to a sigmiodal function (17). Noncompetitive inhibition is exhibited by a variety of purine-, pyrimidine- and pyridine nucleotides (15, 18, 19). It is assumed that these inhibitors bind at a single site, and that the degree of inhibition depends on the total nucleotide concentration, with trinucleotides and dinucleotides being more effective than mononucleotides. Nevertheless the biological significance of these results, and the observation that only aggregates consisting of 16 or 32 subunits are catalytically active, have to be elucidated (16). In physiological conditions the mutation of PRPP-S observed in some patients with gout is manifested in increased activity which is due to decreased sensitivity to feedback inhibition by its intracellular inhibitors (ADP, GDP, 2,3-DPG) (20). This superactive PRPP-S will enhance PRPP formation and, concomitant with activated PRPP-AT, an increased purine synthesis de novo will occur. In patients with superactive PRPP-S, excessive uric acid synthesis could be demonstrated (21). 1.1.2. Regulation of purine nucleotide concentration

The equilibrium ofintracellular purine nucleotides is maintained by the collaboration between purine synthesis de novo, purine interconversion and purine catabolism. The relationship between the actual intracellular concentration of purine nucleotides to the KM-values of enzymes involved in the metabolism of IMP explains the relative rates of this branch point (Figure 4). The KM-values suggest (22-24) that, for example, an increase of IMP secondary to an accelerated de novo purine synthesis will immediately saturate adenylosuccinate synthetase (E.C. 6.3.4.4.) and IMP-dehydrogenase (E.C. 1.2.1.14.). Concomitant activation of cytoplasmic 5'-nucleotidase (5'-PNT, E. C. 3.1.3.5.) will follow and will result in increased hydrolysis of IMP to inosine and subsequent formation of uric

Figure 4 -- Relative affinities of IMP metabolizing enzymes. ADS-S: adenylosuccinate synthetase IMP-DH: IMP dehydrogenase 5'-PNT: 5'-nucleotidase INO: Inosine acid. Since the cytoplasmic 5'-PNT exhibits relatively high KM-values for AMP and GMP, these nucleotides should be protected from degradation and be converted to their trinucleotides. Thus the steady state of adenyland guanyl nucleotides will be maintained in spite of increased de novo synthesis in HGPRT-deficiency and PRPP-S superactivity. 1.1.3. Regulation of adenosine metabolism

Taking into consideration the intracellular substrate concentrations and the kinetic properties of the enzymes involved in adenosine and deoxyadenosine metabolism, the relative rates of the different metabolic routes may be interpreted as follows (Figure 5): Adenosine kinase (AK, E.C. 2.7.1.20.) converts adenosine to AMP and has high affinity but relatively low V.... for adenosine (25). In contrast, adenosine deaminase (ADA, E.C. 3.5.4.4.) exhibits a significantly lower affinity, but a much higher reaction velocity. Furthermore, adenosine might react with homocysteine to form Sadenosyl homocysteine (26, 27). The latter reaction represents a reversal of the usual direction of S-adenosyl homocysteine hydrolase (AHH, E.C. 3.3.1.1.). In contrast to AK and ADA activities determined in lysates of white blood cells under optimal conditions, which might suggest a dominant role of the catabolic route, the affinities of the 3 enzymes mentioned and listed in Figure 5 give first priority within the normal cell to the formation of AMP, since 5 p.mol/L adenosine is present in cells. Probably ADA with its greater working capacity at higher concentrations will act as a "safety-valve" for the breakdown of adenosine. The same could be the case for AHH which should operate only in ADAdeficient cells (28, 26). The metabolism of deoxyadenosine may be important for the explanation of cell-toxicity in ADA-deficient patients (29). The affinity of the deoxyadenosine kinase (dAK) involved in the phosphorylation step is very low compared to ADA (30). Since the intracellular deoxyadenosine concentration is less than 1 ~mol/L, these data suggest that ADA provides the major route of deoxyadenosine metabolism, being responsible for its removal. Deficiency of ADA will lead, therefore, to accumulation of deoxyadenosine and subsequent formation of deoxynucleotides via the kinase reaction (31). Furthermore these enzymes' kinetic data explain the great-

34

NIULLER, KRAUPP AND CHIBA

AMP

KM

KM 77-480

clAMP

ADENOSINE UPTAKE IN HUMAN ERYTHROCYTES 80

KM 4 0 0

2,0

~.

60

S-ADO-HCY <

-AO0

dAD0

./

o 18 °

./: f

pho~pho,vl=hon

40

KM25

KM 25 -30

25 °

dlNO

INO

20

KM-Values : ,umol/I

Figure 5 -- Relative affinities of adenosine and deoxyadenosine metabolizing enzymes. AK: adenosine kinase ADA: adenosine deaminase AHH: S-adenosylhomocysteine hydrolase INO: Inosine S-ADO-HCY: S-adenosylhomocysteine AD: Adenosine TRANSPORT OF PURINECOMPOUNDS

Jp c,~=,l= ,,o.,,,.,,.

:::if(

..............

~ v

I

[ Figure 6--Membrane transport and subsequent intracellular metabolism of purine compounds. B: purine bases NS: purine nucleosides NT: purine nucleotides er accumulation ofdeoxyadenosine than of adenosine in ADA-deficient cells ~32).

~s

3b

eo

1~,o s.to.U=

F i g u r e 7 - - Time dependence of a d e n o s i n e u p t a k e into hum a n e r y t h r o c y t e s at 18 ~ and 25°C respectively. The a l m o s t l i n e a r increase in i n t r a c e l l u l a r radioactivity up to 5 seconds m a i n l y reflects t r a n s p o r t , while the l i n e a r increase a f t e r 30 seconds reflects i n t r a c e l l u l a r metabolic conversion by adenosine kinase. I E x p e r i m e n t s were performed in presence of E H N A 19-erythro-2-hydroxy-3-nonyladeninel to s u p p r e s s a d e n o s i n e d e a m i n a s e activity~.

teins, often called permeases IFigure 6~. Purine bases are trapped intracellularly immediately after passing the membrane by phosphoribosylation. There has been considerable discussion whether transport and phosphoribosylation occur in one step involving group translocation or if phosphoribosylation is subsequent to transport 134-36). Today, the tandem model described in Figure 6 is preferred. Experiments on ATP- a n d / o r PRPP-depleted cells, or on cell lines deficient in one of the purine phosphoribosyltransferases, using incubation times of only a few seconds, revealed that transport was not the rate-limiting step for the uptake of purine bases. On the contrary, the transport process is much faster than subsequent metabolism, and its halfsaturation constant has been determined to be at least one order of magnitude greater than for the subsequent metabolizing enzyme I37). At low extracellular concentrations of purine bases, phosphoribosylation keeps pace with transport, whereas at high extracellular concentrations, the level of free bases in the cell increases due to saturation and relatively low reaction velocity of phosphoribosyltransferases. Furthermore, the initial rates of uptake did not differ between normal and phosphoribosyltransferase-deficient cell lines (37). Nowadays it is assumed that the low KM-system described 138) in earlier studies represents the phosphoribosyltransferase activity rather than the transport process. The number of carriers involved in the uptake of purine bases is still uncertain, but inhibition studies suggest that there are carriers for hypoxanthine and guanine and another one for adenine (33).

1.2. ENZYME DEFECTS AND TRANSPORT

1.2.2. Transport of purine nucleosides 1.2.1. Transport of purine bases Purine bases, like purine nucleosides, pass the cell membrane by facilitated diffusion, which is an enzymatic mechanism where the carrier renders the membrane specifically permeable to certain molecules (33). The whole process is catalyzed by membrane pro-

Purine nucleosides enter cells by the same mechanism just mentioned. Again, the permeases react much faster than the subsequent kinase and deaminase reactions. From kinetic data, it was concluded (39), that at low extracellular concentration of adenosine, overall uptake reflects the kinase activity, whereas at higher

ENZYMOLOGICAL ASPECTS OF DISORDERS IN PURINE METABOLISM

ADO,AMP UPTAKE IN K 5 6 2

35

HL-60

cpm x 10~/10" cells 70

60 3"

:t

50

E 40

30

•

AMP

K 562

•--•

c..,.

30

M St P AC V,r

20

10

5

30

~

I rain

Figure 8 - - Uptake of '"C-AMP into two leukemic cell lines IHL 60 premyelotic and K 562 erythroid) at 37°C in comparison to the respective HC-adenosine uptake. concentrations the uptake rate is limited first by AK and then by HGPRT, since excessive intracellular adenosine will be cleaved by ADA and PNP to hypoxanthine. Figure 7 shows the time dependence of adenosine uptake into human erythrocytes at 18°C and 25°C respectively., Experiments were performed in the presence of 9-erythro-2-hydroxy-3-nonyladenine I EHNAI which potently inhibits ADA activity. Up to five seconds, the graph exhibits an almost linear increase in intracellular radioactivity which is due to the transport process. After that, saturation of adenosine metabolic enzymes occurs. The slight increase of intracellular radioactivity after 30 seconds incubation reflects, under the conditions used, mainly AK activity. The comparison of the kinetic data (see Table 2) of the respective enzymes involved suggests once again that the transport process is not the rate-limiting step, and that AK and HGPRT play the key roles in overall uptake of adenosine into somatic cells t33 L

1.2.3. Transport of purine nucleotides As the membrane in general is impermeable to nucleotides, uptake is dependent on removal of the negatively charged phosphate group. In recent years, it was observed that several lymphoid cell strains were able to accumulate radioactivity after incubation with 14C-AMP. In Figure 8 the uptake of AMP into two human leukemic cell lines, HL-60 (premyelotic), and K562 (erythroid) is shown in comparison. There is strong evidence that the increased uptake of radioactivity into K-562 cells is due to ecto-5'-PNT as demonstrated previously (40, 41) for h u m a n lymphocytes. After cleavage of AMP by ecto-5'-PNT, nucleosides and phosphate moieties might be transported into the cell by means of two different molecules. Whether a metabolic cooper: ation between ecto-5'-PNT and a specific permease exists, or whether the adenosine moiety is transported via the nucleoside carrier, remains unclear. The enzyme defects mentioned in Table 2 might interfere with the uptake processes just described. One could speculate that the deficiency of ecto-5'-PNT in primary agammaglobulinemia might impair the transport of nucleotides in a certain lymphocyte subfraction. On the other hand, the congenital enzyme defects de-

He~

:~, ~,cI Hepal

C~r Per,~ Hepat

ALcohobc No~al¢o~ol,c C,rr~,os,s

Fatly I,ve~

Figure 9 -- Serum guanase activity in a variety of liver diseases. The horizontal bars depict the upper limit of the normal range. Abbreviations used are: ST. P. AC Viral Hep.: status post acute viral hepatitis Chr. Act. Hep.: Chronic active hepatitis Chr. Pets. Hepat.: Chronic persisting hepatitis Units on the vertical axis are given in International Units.

scribed in several forms of hemolytic anemia, and the AMP-DA defect in muscle tissue (42), certainly are followed by decreased levels of intracellular ATP. Under the assumption of metabolic cooperation between transport and subsequent metabolism, the decrease of ATP might certainly impair AK and phosphoribosyltransferase reactions, thus influencing the uptake of purine bases and nucleosides. In fact, AK activities in muscle tissue of patients with AMP-DA deficiency were lower compared to control samples 143).

2. Enzymes in noncongenital diseases Of special importance in this context are purine metabolizing enzymes in malignant cells. In lymphoproliferative diseases ADA, PNP and 5'-PNT might be considered to be of value as diagnostic markers (Table 3}. For example, in acute lymphatic T-cell leukemia (T-ALLI, the ADA activity of peripheral lymphocytes is greatly increased (44, 451, whereas 5'-PNT is almost deficient (46). In contrast, in chronic lymphatic B-cell leukemia {B-CLL), decreased PNP and ADA activities were observed {44, 46). In normal human thymocytes, ADA activity is high as compared to that in peripheral T-lymphocytes (471. On the other hand, 5'-PNT activity is almost lacking in thymocytes (47), whereas peripheral T-lymphocytes have considerable 5'-PNT activity. Therefore, both ADA and 5'-PNT might be interesting as markers of lymphoid cell differentiation. The high ADA and low 5'-PNT activities observed in T-ALL might reflect a maturational arrest of cells of the Tlineage at an early stage of differentiation. The determination of serum guanase (E.C. 3.5.4.3.} activity as a diagnostic tool in liver diseases has been demonstrated. Guanase is mainly restricted to liver, brain and kidney (48} and catalyzes the conversion of guanine to xanthine. Increased serum guanase activities (Figure 9) were observed in a variety of liver diseases. When we compared the diagnostic value of the guanase test with that of parameters such as transaminases, alkaline phosphatase and gammaglutamyltransferase, guanase was shown to be one of the most sensitive indicators of nearly all forms of liver diseases (49}.

36

MI~LLER, KRAUPP AND CHIBA TABLE 3 Lymphocyte Adenosine Deaminase IADA) Activity In Leukemia nmol/1061ymphocytes/h Median

Acute Lymphatic Leukemia IALL) T-ALL common-ALL unclassifiable Chronic Lymphatic Leukemia Controls

3. F u t u r e

5. Johnson, S. M., North, M. E., Asherson, G. L., Allsop, J., Watts, R. W. E.. and Webster, A. D. B.: Lymphocyte purine 5'-nucleotidase deficiency in primary hypogammaglobulinemia. Lancet 1, 168-171 (1977). 6. Jacob, F., and Monod, H.: Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318-356 c 1961 r.

696 114 133 192 324

developments

As already mentioned, structural alterations in an e n z y m e m i g h t lead to altered behaviour towards its physiological regulators. In this context an i n t e r e s t i n g suggestion has recently been made t h a t d e r a n g e d control of liver A M P d e a m i n a s e m i g h t be the cause of an increased b r e a k d o w n of adenine nucleotides (50). This m i g h t result in increased production of uric acid. Indeed, evidence was obtained in a liver biopsy from a h y p e r u r i c e m i c patient t h a t A M P d e a m i n a s e m i g h t be active u n d e r in vivo conditions, thus c a u s i n g the observed h y p e r u r i c e m i a 151 ~. Besides, changes of e n z y m e activities of purine metabolism in several muscle diseases were described (HGPRT, 5'-PNT, A D A ) ( 5 2 - 5 5 1 . However, before conclusions can be d r a w n on the usefulness of these p a r a m eters for diagnosis, more detailed studies have to be done on the pathophysiology of purine metabolism in muscle cells. Evidence has a c c u m u l a t e d t h a t purines (adenosine, ATP) play a role in neurophysiological processes (56). Undoubtedly, e n z y m e s acting on these purines will be i m p o r t a n t in the regulation of these processes. In conclusion, it should be stated t h a t e n z y m e s involved in purine metabolism can be considered as par a m e t e r s in the diagnosis of a n u m b e r of inborn errors, m a l i g n a n c i e s and as indicators of liver dysfunction. It s e e m s probable t h a t f u r t h e r f u n d a m e n t a l e n z y m a t i c studies on purine metabolism m i g h t increase the importance of this p a t h w a y and its e n z y m e s for the clinical enzymologist. References

1. Watts, R. W. E., Watts, J. E. M. and Seegmiller, J. E.: Xanthinoxidase activity in human tissues and its inhibition by allopurino114-hydroxypyrazolo 13,4-d~pyrimidine). J. Lab. Clin. Med. 66. 688-697 (1965). 2. Rijksen, G., Staal, G. E. J. van der Vlist, M. J. M., Beemer, F. A., Troost, J., Gutensohn, W., van Laarhoven, J. P. R. M., and de Bruyn, C. H. M. M.: Partial hypoxanthineguanine phosphoribosyltransferase deficiency with full expression of the Lesch-Nyhan syndrome. Hum. Genet. 57, 39-47 (1981~. 3. De Bruyn, C. H. M. M.: Hypoxanthine-guanine phosphoribosyl transferase deficiency. Hum. Genet. 31, 127-150 (1976). 4. Mfiller, M. M., and Kuzmits, R.: Prfinatale Diagnostik von Enzymdefekten des Purinstoffwechsels. In: E. Kaiser and M. M. Mfiller: Prdnatale Diagnostik und Heterozygotennachweis, pp. 61-77, Facultas Verlag, Vienna, 1979.

7. Schimke. R. T., and Doyle, D.: Control of enzyme levels in animal tissues. Ann. Rev. Bioclwm. 39, 929-976 11970). 8. Wood. A. W., Becker, M. A., and Seegmiller, J. E.: Purine nucleotide synthesis in lymphoblasts cultured fi'om norreal subjects and a patient with the Lesch-Nyhan syndrome. Biochem. Genet. 9, 261-274 (1973L 9. Seegmiller, J. E.. Klinenberg, d. R., Miller, J., and Watts, R. W. E.: Suppression of glycine-~r'N incorporation into urinary uric acid by adenine-8-~~C in normal and gouty subjects. J. Clin. Invest. 47, 1193-1203 (1968). 10. Seegmiller, J. E., Rosenbloom, F. M.. and Kelley, W. N.: Enzyme defects associated with a sex-linked neurological disorder and excessive purine synthesis. Science 155, 1682-1684 11967~. 11. Holmes, E. W., McDonald. J. A., McCord. J. M., Wyngaarden, J. B., and Kelley, W. N.: Human glutamine phosphoribosylpyrophosphate amidotransferase: kinetic and regulatory properties. J. Biol. Chem. 248, 144-150 11973~. 12. Holmes, E. W.. Wyngaarden, J. B., and Kelley, W. N.: Human glutamine phosphoribosylpyrophosphate amidotransferase: two molecular forms interconvertible by purine ribonucleotides and phosphoribosylpyrophosphate. J. Biol. Chem. 248, 6035-6040 ~1973). 13. Henderson, J. F., Brox, L. W., Kelley. W. N., Rosenbloom, F. M., and Seegmiller, J. E.: Kinetic studies of hypoxanthine-guanine phosphoribosyl transferase. J. Biol. Chem. 243. 2514-2522 11968~. 14. Nelson, D. J., Bugge, C. J. L., Krasny, H. C., and Elion, G. B.: Formation of nucleotides of 16-~~C) oxipurinol in rat tissues and effects on uridine nucleotide pools. Biochem. Pharmacol. 22, 2003-2022 119731. 15. Fox, I. H., and Kelley, W. N.: Human phosphoribosylpyrophosphate synthetase: kinetic mechanism and endproduct inhibition. J. Biol. Chem. 247, 2126-213111972). 16. Meyer, L. J., and Becker, M. A.: Human erythrocyte phosphoribosylpyrophosphate synthetase. Dependance of activity on state of subunit association. J. Biol. Chem. 252, 3919-3925 11977). 17. Hershko, A., Razin, A., and Mager, J.: Regulation of the synthesis of 5-phosphoribosyl-l-pyrophosphate in intact red blood cells and cell-free preparations. Biochim. Biophys. Acta 184, 64-76 I1969). 18. Switzer, R. L., and Sogin, D. C.: Regulation and mechanism of phosphoribosylpyrophosphate synthetase. V. Inhibition by end-products and regulation by adenosine diphosphate. J. Biol. Chem. 248, 1063-1073 11973). 19. Olszowy, J., and Switzer, R. L.: Specific repression ofphosphoribosylpyrophosphate synthetase by uridine compounds in Salmonella typhimurium. J. Baeteriol. 110, 450-451 {1972). 20. Zoref, E., De Vries, A., and Sperling, O.: Mutant feedbackresistant phosphoribosylpyrophosphate synthetase associated with purine overproduction and gout, phosphoribosylpyrophosphate and purine metabolism in cultured fibroblasts. J. Clin. Invest. 56, 1039-1099 (1975). 21. Mfiller, M. M., and Frank, O.: Lipid and purine metabolism in benign symmetric lipomatosis. Adv. Exp. Med. Biol. 41B, 509-516 t1974). 22. Van der Weyden, M., and Kelley, W. N.: Human adenylosuccinate synthetase. Partial purification, kinetic and regulatory properties of the enzyme. J. Biol. Chem. 249, 7282-7289 (1974). 23. Holmes, E. W., Pehlke, M. and Kelley, W. N.: Human IMP

ENZYMOLOGICAL ASPECTS OF DISORDERS IN PURINE METABOLISM

24. 25.

26. 27.

28. 29.

30.

31.

32.

33.

34.

35. 36. 37.

38.

dehydrogenase. Kinetics and regulatory properties. Biochim. Biophys. Acta 364, 208-217 11974). Itoh, R., Mitsui, A., and Tsushima, K.: 5'-Nucleotidase of chicken liver. Biochim. Biophys. Acla 146, 151-159 11967). Schnebli, H. P., Hill, D. L., and Bennett, L. L. Jr.: Purification and properties of adenosine kinase from human tumor cells of type H. Ep. No. 2, J. Biol. Chem. 242, 1997-2004 (1967). Hershfield. M. S., and Kredich, N. M.: A mechanism for adenosine IADO~ cytotoxicity in adenosine deaminase (ADAI deficiency. Clin. Res. 26, 329A. 11978). Richards, H. H., Chiang, P. K., and Cantoni, G. L.: Adenosylhomocysteine hydrolase. Crystallization of the purified enzyme and its properties. J. Biol. Chem. 253, 4476-4480 t 1978). Hershfield, M. S.: Human cytoplasmic adenosine binding protein: Identification as S-adenosyl-homocysteinase. Fed. Proc. 37, 1466 )1978). Giblett, E. R., Anderson, J. E,, Cohen, F., Pollara, B., and Meuwissen, J. J.: Adenosine-deaminase deficiency in two patients with severely impaired cellular immunity. Lancet 2, 1067-1069 11972). Carson, D. A., Kaye, J., and Seegmiller, J. E.: Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: Possible role of nucleoside kinaselsL Proc. Natl. Aead. Sei. {USA J 74, 5677-5681 11977). Coleman, M. S., Donofrio, J., Hutton. J. J., Hahn, L.. Daoud, A., Lampkin, B,, and Dyminski, J.: Identification and quantitation of adenine deoxynucleotides in erythrocytes of a patient with adenosine deaminase deficiency and severe combined immunodeficiency. -J. Biol. Chem. 253, 1619-1626 (1978). Cohen, A., Hirshhorn, R., Horowitz, S. D., Rubinstein, A.. Polmar, S. H., Hong, R., and Martin, D. W., Jr.: Deoxyadenosine triphosphate as a potentially toxic metabolite in adenosine deaminase deficiency. Proe. Natl. Aead. Sei. tUSAJ 75, 472-476 11978). Wohlhueter, R. M., and Plagemann, P. G.: The roles of transport and phosphorylation in nutrient uptake in cultured animal cells. Internat. Rev. Cytology 64. 171-240 (1980L Quinland, D. C., and Hochstadt, J.: Group translocation of the ribose moiety of inosine by vesicles of plasma membrane from 3T3 cells transformed by Simian virus 40. ,]. Biol. Chem. 251,344-354 11976L Hawkins, R., and Berlin, R.: Purine transport in polymorphonuclear leukocytes. Biochim. Biophys. Acta 173, 324-337 ¢1969). Mfiller, M. MI, Kraupp, M., and de Bruyn, C. H. M. M.: Purine base transport in normal and mutant erythrocytes. Hum. Hered. 29, 118-123 ( 1979 ). Marz, R., Wohlhueter, R. M., and Plagemann, P. G. W.: Purine and pyrimidine transport and phosphoribosylation and their interaction in overall uptake by cultured mammalian cells. A re-evaluation. J. Biol. Chem. 254, 2329-2338 (1979). Mfiller, M. M., and Falkner, B.: Uptake of hypoxanthine in human erythrocytes. Adv. Exp. Med. Biol. 76B, 131-138 11977).

37

39. Lum, C., Marz, R., Plagemann, P. G. W., and Wohlhueter, R.: Adenosine transport and metabolism in mouse leukemia cells, in canine thymocytes and peripheral blood leukocytes. J. Cellul. Physiol. 101, 173-200 119791. 40. Edwards, N. L., Gelfand, E. W., Burk, L., Dosch, H. M., and Fox, I.: Distribution of 5'-nucleotidase in human lymphoid tissues. Proc. Natl. Acud. Sci. ¢USA J 76, 3474-3476 11979). 41. Fleit, H., Conklyn, M., Stebbins, R. D., and Silber, R.: Uptake of adenosine fi'om AMP by human lymphocytes. J. Biol. Chem. 250, 8889-8892 11975~. 42. Fishbein, W. N., Armbrustmacher, V. W., and Griffin, J. L.: Myoadenylate deaminase deficiency: a new disease of muscle. Science 200, 545-548 11978). 43. Valentine, W. N., Paglia, D. E., Tartaglia, A. P., and Gilsanz, F.: Hereditary hemolytic anemia with increased red cell adenosine deaminase 145- to 70-fold~ and decreased adenosine triphosphate. Science 195, 783-785 11977). 44. Ludwig, H., Kuzmits, R., Pietschmann, H., and Mtiller, M. M.: Enzymes of the purine interconversion system in chronic lymphatic leukemia: decreased purine nucleoside phosphorylase and adenosine deaminase activity. Blut 39, 309-315 ~1979L 45. Ludwig. H.. Winterleitner, H., Kuzmits, R.. and Mtiller, M. M.: Adenosine deaminase and purine nucleoside phosphorylase in acute and chronic lymphatic leukemia. Adv. Exp. Med. Biol. 122B, 333-337 ~1980L 46. Quagliata, F., Faig, D., Conklyn, M., and Silber, R.: Studies on the lymphocyte 5'-nucleotidase in chronic lymphocytic leukemia, infectious mononucleosis, normal subpopulations, and phytohemagglutinin-stimulated cells. Cancer Res. 34, 3197-3202 (1974~. 47. Van Laarhoven, J. P. R. M., and de Bruyn, C. H. M. M.: Personal communication. 48. Kuzmits, R., Stemberger, H., and Mtiller, M. M,: Guanase from human liver - - purification and characterization. Adv. Exp. Med. Biol. 122B, 183-188 (19801. 49. Kuzmits, R., Seyfried, H., Wolf, A.. and Mtiller, M. M.: Evaluation of serum guanase in hepatic diseases. Enzyme 25, 148-152 119801. 50. Hers, H.-G., and van den Berghe, G.: Enzyme defect in primary gout. Lancet 1,585-586 11979L 51. Van den Berghe, G.: In preparation. Lancet 11981L 52. Mfiller, M. M., Kuzmits, R., Frass, M., and Mamoli, B.: Purine metabolism of erythrocytes in myotonic dystrophy. J. Neurol. 223, 59-66 119801. 53. Neerunjun, J. S., Allsop, J., and Dubowitz, V.: Hypoxanthine-guanine phosphoribosyltransferase activity of blood and muscle in Duchenne dystrophy. Muscle and Nerve 2, 19-23 (1979~. 54. Kar, N. C., and Pearson, C. M.: Nucleotidase activity of normal and dystrophic human muscle. Proc. Soc. Exp. Biol. Med. 143, 1125-1126 )1973). 55. Kar, N. C., and Pearson, C. M.: Muscle adenylic acid deaminase activity. Neurology 23, 479-482 (1973). 56. Burnstock, G.: The purinergic nerve hypothesis. In: Purine and Pyrimidine Metabolism, Ciba Foundation Symposium 48, pp. 295-314, Elsevier, Amsterdam, Oxford, New York, 1977.