Erythrocyte phosphoribosylpyrophosphate concentrations in heterozygotes for hypoxanthine-guanine phosphoribosyltransferase deficiency

Erythrocyte Phosphorihosylpyrophosphate Concentrations in Heterozygotes for Hypoxanthine-Guanine Phosphorib;osyltransferase Deficiency Ross B. Gordon,

Lombert Thompson,

tleterozygotes for the deficiency of hypoxonthine-guonine phosphoribosyltransferose (HGPRT) demonstrate either normal erythrocyte HGPRT activities in families in which the hemizygote shows the leschNyhon syndrome, or o range of erythrocyte HGPRT activities between 20% of normal and completely normal values in families in which the mutation results in urote overproduction with few or no nourologic Phosphoribosylpyrophosphote signs. (PRPP) concentmtions in lrythrocytes from heterozygotes for HGPRT deficiency were

and Bryan T. Emmerson

shown to correlate inversely vith HGPRT activities and directly with APRT activities in erythrocyte lysates. Since no evidence of increased sy,nthesis of PRPP war obtained, these elevated PRPP concentrations con best be attributed to reduced utilizotion of PRRP in the HGPRT-catalyzed reaction. As shown in hemizygotes, the i& creased PRPP ,concentrotions in these heterozygotes could result in stabilization of the APRT enzyme and elevotion of APRT activities.

D

EFICIENCY OF hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity is regularly characterized by overproduction of mate, the more severe deficiencies being associated with the Lesch-Nyhan syndrome of mental deficiency, choreo-athetosis and compulsive self-mutilation, while the less severe deficiencies are associated either with no neurologic manifestations or with a spectrum ranging up to those of the .Lesch-Nyhan syndrome.‘-3 Heterozygotes for the Lesch-Nyhan syndrome have usually shown normal HGPRT activities in erythrocyte lysates, although cultures of skin fibroblasts have shown them to be mosaics of cells with either,normal,or deficient HGPRT activity.’ However, heterozygotes for the less severe HGPRT deficiency states have often shown a range of HGPRT activities between 22% of normal and completely normal values in erythrocyte lysates, although some heterozygotes from the same family have demonstrated normal HGPRT activities.s Such intermediate HGPRT activities were not seen in heterozygotes for the LeschNyhan syndrome. It has recently been proposed that the concentration and rate of synthesis, of the high-energy metabolite phosphoribosylpyrophosphate (PRPP) plays a critical role in the regulation of purine biosynthesis de novo.6 Hershko et al.’ found increased in vitro formation of PRPP in the erythrocytes of some gouty paFrom rhe University of Queensland Department of Medicine at the Princess Alexandra Hospital, Brisbane, Australia. Presenred in part at the International Symposium on Purine Metabolism in Man, Tel Aviv, Israel, June I&Zt.l973. Receivedfor publication January 28. 1974. Supported chiefly by the National Health and Medical Research Council of Ausiralia. Reprinr requests should be addressed to Dr. B. T. Emmerson. Princess Alexandra Hospital, Woolloongabba Q.4102. Australia. 0 1974 by Grune & Stratton. Inc.

Metabolism, Vol. 23, No. 10 (October), 1974

921

922

GORDON,

THOMPSON,

AND

EMMERSON

tients. These authors further suggested that the increase in the rate of purine base uptake found in these patients was attributable to the enhanced PRPP formation. In addition, it has been found that patients with both severe and moderate degrees of HGPRT deficiency have elevated erythrocyte PRPP concentrations,6*8 a feature rarely found in erythrocytes from patients with gout who had normal HGPRT activity. These elevated erythrocyte PRPP concentrations in HGPRT-deficient subjects were attributed to diminished utilization of this compound by the HGPRT-catalyzed reaction.* Since PRPP concentrations had been shown to be abnormal in erythrocytes from HGPRT-deficient hemizygotes, it was decided to investigate the corresponding situation in heterozygotes. MATERIALS

AND METHODS

The concentration of PRPP in erythrocytes was measured essentially as described by Sperling et a1..9 with PRPP being the limiting factor in the conversion of a purine base to its nucleotide and utilizing a phosphoribosyltransferase activity already present in the erythrocyte. Further PRPP synthesis during the assay was inhibited by 2,3-diphosphoglyceric acid (2.3, DPG). In subjects with normal HGPRT and APRT activity, this method yields comparable values for PRPP concentration when either the APRT or the HGPRT activity is used. Both methods yield a linear response to increasing PRPP concentration.’ Since some of the heterozygotes studied had low HGPRT activity, the PRPP assay was undertaken using the adenine phosphoribosyltransferase (APRT) activity present in the hemolysate with ‘*C-adenine as the second substrate. Plasma, leukocytes, and platelets were removed from freshly drawn heparinized blood by centrifugation and the erythrocytes washed twice with cold saline. Lysis of erythrocytes was effected by adding 100 ~1 of packed cells to 200 pl of cold distilled water and subjecting the mixture to two cycles of freezing and thawing. The lysate was spun at 3000 rpm for IO-20 min at 4°C to remove stroma. Hemolysate (50 ~1) was incubated in the presence of 5.5 pmoles of Tris buffer, pH 7.4, 0.5 rmoles MgCI,, 0.05 pmoles adenine-8-‘*C (SA IO r.&i/rmole, Radiochemical Centre, Amersham, used without further purification), and I rmole of 2.3 DPG in a final volume of 100 ~1. Less than I% of the adenine was converted to nucleotide. After incubation for 60 min at 37’C, the reaction was stopped by the addition of 50 ~1 of solution containing 2 Mmoles of EDTA and 0.05 pmoles of carrier nucleotide, followed by immediate freezing in a dry ice-alcohol bath. Aliquots of 10 ~1 of the reaction mixture were then spotted on Whatman 3-MM paper and subjected to high-voltage electrophoresis (3000 V for 30 min) in 0.05 M borate buffer, pH 8.9, containing 0.02 M KCI and 0.001 M EDTA. The radioactive adenylic acid spots were located using uv light, cut out, and counted in a Nuclear Chicago Mark I liquid scintillation counter at 70’4-75% efficiency using external standardization. The scintillation fluor consisted of PPO (4 g) and POPOP (0.1 g) per liter of toluene, to each liter of which 500 ml of Triton-Xl00 was added. PRPP synthetase activity was assayed by the ribose-5-phosphate-dependent conversion of ‘*C-ATP (Radiochemical Centre, Amersham) to ‘*C-AMP.” PRPP generating capacity was assessed by incubating erythrocytes in a series of phosphate-glucose buffers in which the phosphate concentration varied between 5 and 50 mM”: after washing with cold saline, the erythrocytes were lysed and their PRPP content measured.‘* Assays for HGPRT and APRT activities were carried out on lysates that had been dialyzed against 0.001 M Tris buffer, pH 7.0, for I6 hr. as described by Emmerson et al.’ Plasma urate levels were determined by the uricase method of Liddle et al.” Protein was estimated by the method of Lowry et al.,‘* using bovine serum albumin as the standard. The heterozygotes studied were asymptomatic, and none was receiving any drug, including allopurinol, or other substance known to affect urate metabolism or erythrocyte PRPP concentration.‘* The hemizygotes for the four families have been described in detail, those from family I demonstrating the Lesch-Nyhan syndrome (J.W. and R.W.3), while those from the other families demonstrated less severe HGPRT deficiency states (family 2, G.L.; family 3, F.B. and L.B.; family 4, A.C. and C.C.3).

HYPOXANTHINE-GUANINE

PHOSPHORIBOSYLTRANSFERASE DEFICIENCY

923

Table 1. Erythrocyte PRPPConcentrotionr ond Phosphoriboryltronrferore Activities in Heterorygoter for HGPRT-Deficiency PRPP nmolcr/ml

Normal females

HGPRT Activity nmokr/hr/mg

APRT Activity nmoler/hr/mg

Eryihrocytcs

Protein

Protein

11.6+2.8(18)

94 f 7.7 (24)

20.1 f 2.5 (22)

Plasma Urote mg/lOO

4.2 f

ml

1.2

(Mean f SD) Heterozygote Family 1 8.W.

9.6

89.0

21.1

4.7

E.D.

10.1

85.0

19.0

3.9

E.G.

6.0

88.0

18.6

5.5

R.B.

8.4

75.0

18.1

6.5

25.2

10.1

31.5

4.1

Family

2

R.L. Family 3 LB.

10.1

71.0

25.5

4.8

C.B.

14.4

52.0

26.5

6.6

M.8.

19.4

28.0

28.4

4.3

V.B.

22.0

17.7

28.2

4.7

15.3

35.5

28.5

7.3

Family 4 ML.

RESULTS

Table I shows the erythrocyte PRPP concentrations for normal females, for heterozygotes for the Lesch-Nyhan syndrome (family I), and for the moderate HGPRT-deficiency syndrome (families 2, 3, and 4); it also shows the HGPRT and APRT activities in erythrocyte lysates. It will be seen that erythrocyte PRPP concentrations were normal in heterozygotes for the Lesch-Nyhan syndrome but were elevated in some of the heterozygotes from families affected by moderate HGPRT deficiency. The greater the depression of the erythrocyte HGPRT activity, the greater appeared to be the concentration of PRPP. Thus a significant negative correlation (r = -0.92, p < 0.001) was demonstrated between the erythrocyte PRPP concentration and the HGPRT activity (Fig. l), thereby supporting the suggestion that the PRPP concentration in erythrocytes is influenced by the activity of the HGPRT enzyme. This correlation was not seen if family 1 was viewed alone, but it should be noted that this was the only family in which the hemizygote demonstrated the Lesch-Nyhan syndrome and the only family in which the heterozygotes showed either normal or near normal HGPRT and APRT activities in erythrocytes.5 A significant positive correlation (r = 0.89, p < 0.001) was also demonstrable between erythrocyte APRT activity and PRPP levels for these heterozygotes (Fig. 2). Such a relationship was not seen constantly in every subject, and L.B. shows a marginally elevated APRT activity with a normal erythrocyte PRPP concentration. It may be noted, however, that on different occasions over a 2-yr period the APRT activity in L.B. was sometimes clearly within the normal range and at other times clearly elevated. The erythrocyte PRPP concentrations found in normal subjects were similar to those reported by Sperling et aL9 using the present method but were slightly

r = -0.92

P<

I

I

20

40

HPRT nmolcs

0.001

I

I

80

60

I

I

100

120

be1. The relationship the PRPP concentration and HGPRT activity of erythrocyte lysates from ten heterozygotes for HGPRT deficiency. Fig.

tween

ACTIVITY /hr/mg

r= 0.89 P
Fig. 2. The relationship between the APRT activity and PRPP concentration of erythrocyte lysates as found in ten heteroxygotes for HGPRT deficiency. 9324

I

I

I

I

I

1

1

4

8

12

16

20

24

nmoler/ml

PRPP Erythrocytet

I 28

HYPOXANTHINE-GUANINE

925

DEFICIENCY

PHOSPHORlBOSYLTRANSFERASE

Normal

(6)

l

HeterozyBotes

PHOSPHATE

R.L.

A

V.B.

o

C.B.

o

mM

fig. 3. The effect of phosphate concentration on the amount of PRPP produced in red cells during 60 min incubation in phosphate-glucose buffer in six normal subjects (mean f SD) and three heterozygotes for HGPRT deficiency.

This was attributed to the fact that this higher than previously described. 6;1~12 particular method9 does not involve either heating or charcoal adsorption, both of which procedures in our hands result in a reduction of PRPP content. We also found the PRPP concentration in normal females (Table 1) to be significantly higher than that in normal males (8.0 + 1.0; n = 13),15 a finding previously described using a different method of PRPP measurement.‘6 Since mutants of PRPP synthetase have been described as a cause of elevated it was considered important to establish erythrocyte PRPP concentrations,“,” the absence of such mutations in the heterozygotes for the moderate HGPRT deficiency. The PRPP synthetase activities of erythrocyte lysates from three of these heterozygotes (R.L., 34.2; V.B., 34.6; C.B., 45.2 nm/hr/mg) were found not to be different from the activities of lysates from normal control subjects [41.2 f 10.2 (n = 6)]. In addition, the PRPP generating capacity of the erythrocytes from these three heterozygotes was normal at several different phosphate concentrations (Fig. 3). It therefore appears unlikely that the high levels

926

GORDON,

of intracellular PRPP found in the heterozygotes of PRPP. No correlation was found between erythrocyte corresponding plasma urate concentration.

THOMPSON,

AND EMMERSON

are due to increased PRPP

concentrations

synthesis and the

DISCUSSION

The data presented show that erythrocyte PRPP concentrations in heterozygotes for the moderate HGPRT deficiency reflect the activity of the HGPRT enzyme in erythrocytes. Since PRPP synthetase activity is normal, it seems likely that the PRPP content becomes elevated due to a decrease in its consumption by the HGPRT reaction. A significant correlation was also found between erythrocyte APRT activity and PRPP concentrations. Rubin et al. I8 have shown that the increased APRT activity in erythrocyte lysates from HGPRT-deficient children was due to prolongation of the half-life of APRT, which they attributed to increased APRT enzyme stability, while Greene et a1.i9 attributed this increased stability to the increased content of PRPP, which they demonstrated in HGPRTdeficient erythrocytes. Furthermore, immunologic studies recently reportedZo have demonstrated that erythrocytes from Lesch-Nyhan patients and from normal subjects contain the same amount of APRT enzyme protein. Such evidence of immunologic identity appears more consistent with the postulate of stabilization of the APRT enzyme by endogenous metabolites rather than the result of an alteration in the enzyme structure. Our findings of an increased PRPP concentration in heterozygotes for HGPRT deficiency, taken in conjunction with similar findings in hemizygotes for HGPRT deficiency,19 provide evidence that the metabolite responsible for the increased APRT activity in these cases is PRPP and provide further support for an explanation based on stabilization of the APRT enzyme. Evidence against stabilization by PRPP has been reported by Kelley,*’ who found that cultured fibroblasts from patients with HGPRT deficiency consistently showed normal activity of the APRT enzyme, even though such fibroblasts demonstrated increased intracellular concentrations of PRPP. A similar lack of correlation between plasma urate concentration and erythrocyte PRPP concentration has also been reported by Fox and Kelley.6 However, it has been demonstrated that abnormalities of de novo purine synthesis may coexist with normal plasma urate concentrations, and several of the heterozygotes studied have shown this feature. 22 If the PRPP concentration in erythrocytes reflects in any way the levels in other tissues, such increased concentrations may form part of the biochemical basis for the abnormal urate metabolism demonstrated in heterozygotes for the moderate HGPRT deficiency. ACKNOWLEDGMENT The Life Insurance

Medical

Research

Fund of Australia

contributed

to the equipment

used.

REFERENCES 1. Seegmiller JE, Rosenbloom FM, Kelley WN: Enzyme defect associated with a sexlinked human neurological disorder and excessive purine synthesis. Science 155: 1682, 1967

2. Kelley WN, Greene ML, Rosenbloom FM et al.: Hypoxanthine-guanine phosphoribosyltransferase deficiency in gout. Ann Intern Med 70: 155, 1969

HYPOXANTHINE-GUANINE

PHOSPHORIBOSYLTRANSFERASE DEFICIENCY

3. Emmerson BT, Thompson L: The spectrum of hypoxanthine-guanine phosphoribosyltransferase deficiency. Q J Med 42:423, 1973 4. Migeon BR, Kaloustian VMD, Nyhan WL et al.: X-linked hypoxanthine-guanine phosphoribosyltransferase deficiency: Heterozygote has two clonal populations. Science 160:425, 1968 5. Emmerson BT, Thompson CJ, Wallace DC: Partial deficiency of hypoxanthineguanine phosphoribosyltransferase: Intermediate enzyme deficiency in heterozygote red cells. Ann Intern Med 76:285, 1972 6. Fox IH, Kelley WN: Phosphoribosylpyrophosphate in man: Biochemical and clinical significance. Ann Intern Med 74:424, 1971 7. Hershko A, Hershko C, Mager J: Increased formation of %phosphoribosyl-l-pyrophosphate in red blood cells of some gouty patients. Isr J Med Sci 4:939, 1968 8. Green ML, Seegmiller JE: Elevated erythrocyte phosphoribosylpyrophosphate in X-linked uric aciduria: Importance of PRPP concentration in the regulation of human purine biosynthesis. J Clin Invest 48:32a, 1969 9. Sperling 0, Eilam G, Persky-Brosh S et al.: Simpler method for the determination of 5-phosphoribosyl-I-pyrophosphate in red blood cells. J Lab Clin Med 79:1021, 1972 IO. Fox IH, Kelley WN: Human phosphoribosylpyrophosphate synthetase. J Biol Chem 24615739, 1971 Il. Sperling 0, Boer P, Persky-Brosh S et al.: Altered kinetic property of erythrocyte phosphoribosylpyrophosphate synthetase in excessive purine production. Rev Eur Etud Clin Biol 17:24, 1972 12. Fox IH, Wyngaarden JB, Kelley WN: Depletion of erythrocyte phosphoribosylpyrophosphate in man-a newly observed effect of allopurinol. N Engl J Med 283: 1177, 1970 13. Liddle L, Seegmiller JE, Laster L: The

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enzymatic spectrophotometric method for determination of uric acid. J Lab Clin Med 54:903, 1959 14. Lowry OH, Rosebrough NJ, Farr AL et al.: Protein measurement with the Folin-phenol reagent. J Biol Chem 193:265, 1951 15. Emmerson BT, Gordon RB, Thompson L: Adenine phosphoribosyltransferase deficiency in a female with gout, in Sperling 0, Rivlin G (eds): Purine Metabolism in Man. New York, Plenum, 1974, p 327 16. Meyskens FL, Williams HE: Concentration and synthesis of phosphoribosylpyrophosphate in erythrocytes from normal, hyperuricemic and gouty subjects. Metabolism 20~737, 197 I 17. Becker MA. Meyer LJ, Wood AW et al.: Purine overproduction in man associated with increased phosphoribosylpyrophosphate synthetase activity. Science 179: 1123, 1973 18. Rubin CS, Balis ME, Piomelli S et al.: Elevated AMP pyrophosphorylase activity in congenital IMP pyrophosphorylase deficiency (Lesch-Nyhan disease). J Lab Clin Med 74732. 1969 19. Greene ML, Boyle JR, Seegmiller JE: Substrate stabilisation: genetically controlled reciprocal relationship of two human enzymes. Science 167:887, 1970 20. Yip LC, Dancis J, Balis ME: Immunochemical studies of AMP-pyrophosphate phosphoribosyltransferase from normal and Lesch-Nyhan subjects. Biochim Biophys Acta 293:359, 1973 21. Kelley WN: Studies on the adenine phosphoribosyltransferase enzyme in human fibroblasts lacking hypoxanthine-guanine phosphoribosyltransferase. J Lab Clin Med 77:33, 1971 22. Emmerson BT, Wyngaarden JB: Purine metabolism in heterozygous carriers of hypoxanthine-guanine phosphoribosyltransferase deficiency. Science 166: 1533, 1969