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Evaluation of the role of 5-phosphoribosyl-α-1-pyrophosphate synthetase in congenital hyperuricemia and gout: A simple isotopic assay and an activity stain for the enzyme
Evaluation
of
the
Role
phate Synthetase Gout: A Simple
of
Department
-Pyrophos-
in Congenital Hyperuricemia and Isotopic Assay and an Activity
Stain MARTIN
5-Phosphoribosyl-a-1
for the Enzyme
G. JOHNSON, AND ROBERT
of Bioc.hc.mistr!y.
SIMON ROSENZWEIG L. SWITZER
Vniversitl/
of lliinoi.c,
Urbana.
2llinoi.r
6!ROl
AND
MICHAEL Department
A. BECKER of Medicine,
University California Received
J. EDWIN
AND
of Californk 92037
October
SEEGMILLER at Sal& Diego,
La Jolla,
29, 1973
INTRODUCTION
Among the mechanisms that have been suggested for the excessive uric acid production of a significant proportion of patients with primnry metabolic gout are defects that lead to excessive concentrations of phosphoribosylpyrophosphate (PRPP) , a substrate for the first step of purine biosynthesis de no~o (1). This reaction, catalyzed by PRPP:glutamine amidotransferase, is sensitive to feedback inhibition by purine nucleotides; these nucleotides, while probably binding at specific allosteric sites, are kinetically competitive with respect to PRPP (24). Thus, an elevation of PRPP concentrations above normal levels would be expected to increase the rate of purine synthesis not only because of increased availability of a substrate for PRPP : glutamine amidotransferasc, but also as a result of a desensitization of the first enzyme of purine synthesis to feedback control, possibly through a conversion from the less active dimer to a more active monomeric form (4). The view that PRPP concentration may be an important element in excessive uric acid production in man first received experimental support from studies of the Lesch-Nyhan syndrome, an inherited disease characterized by mental retardation, choreoathetosis, self-mutilation, and extreme over-production of uric acid (5). The Lesch-Nyhan syndrome Copyright .4Il rights
266 @ 1974 by Academic Press, Inc. of reproduction in any forln rrserwd.
PRPP
SYNTHETASE
IN
EXCESSIVE
PURINE
PRODUCTION
267
results from a severe deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (6). Erythrocytes, fibroblasts, and lymphoblasts from affected individuals show substantially increased concentrations of the free PRPP (7-10). Subsequently, Sperling et ~2. (11) and Becker et al. (12) have independently reported the occurrence of families with inherited hyperuricemia and gout in which erythrocytes from affected individuals possess elevated PRPP synthetase (EC 2.7.6.1) activity. The fact that the abnormal PRPP synthetase activities discovered by these two groups have very different kinetic properties suggests that a variety of PRPP synthetase defects may be associated with purine overproduction ( 12-14). It is clear, however, that, in many hyperuricemic or gouty individuals with excessive purine production, altered levels of PRPP or of PRPP synthetase activity cannot be detected, at least in erythrocytes (8, 9, 15, 16). Recently, an instance of hemolytic anemia associated with a deficiency of PRPP synthetase has b-en described ( 17). The frequency of occurrence and variety of metabolic disorders resulting from abnormalities in the functioning of PRPP synthetase remains to be fully established. This paper presents a method for sensitive. specific, and relatively simple determination of PRPP synthetase activity in human erythrocyte lysates that is free of some of the problems of other techniques. In addition, an activity stain for PRPP synthetase that has proven useful in studying electrophoretic behavior of normal and mutant enzymes is presented. These methods should prove useful in diagnosis and study of metabolic disorders that may involve PRPP concentrations and PRPP synthetasc activity. MATERIALS
AND METHODS
Materials. Adenosine triphosphate, ribose-5-phosphate, creatine phosphate, TPN”, phenazine methosulfate, and nitrobluetetrazolium were purchased from Calbiochem or Sigma. PRPP, dimagnesium salt, was the product of P-L Biochemicals, Inc. ATP-y-“‘P was prepared by the exchange procedure of Glynn and Chappel ( la), purified, and characterized as previously described ( 19). Hexokinase, glucose-6-phosphate dehydrogenase, creatine phosphokinase, and the enzymes used in the synthesis of ATP-Y-“~P were highly purified preparations obtained from Sigma. The mixture of orotidylic pyrophosphorylase and orotidylic decarboxylase used for PRPP assays by the orotate removal method (20) was a crude preparation from yeast sold by P-L Biochemicals, Inc. Preparation of Hemolysates. Blood was collected by venipuncture into heparinized tubes, which were kept on ice. Erythrocytes were separated from the plasma by centrifugation at 1900 X g for 15 min and washed three times with 4 vol of cold 0.9%NaCI, buffered to pH 7.4 by addition
of 1 M tris-Cl, pIf 7.4. The packed cells were stored at - 10” until use. Hemolysates were prepared from frozen crythrocytes by thawing the cells in 3 vol of cold 1 rnM EDTA, pH 7. After standing at O-2” for 15 min, lysis of the erythrocytes was complete. Partial Purification of PRPP Synthetase for Activity Staining. The activity stain has not been successfully applied to crude hemolysates; hence, the PRPP synthetase used in activity staining was partially (5090-fold) purified from 10-15 ml of packed, washed cells using a slight modification of the DEAE-cellulose batch elution step previously described by Fox and Kelley (21). Th e volumes of solutions used we’-e all l/20 of the volumes listed, and intermediate washing of the DEAEcellulose with 3 mM sodium phosphate-50 rnM KC1 buffer was omitted since it did not improve the purification significantly. Assa!y for PRPP Synthetase. The assny for PRPP synthetase is based on enzymic transfer of radioactivity from ATP-y-“‘P to PRPP and/or its acid degradation products. The unreacted ATP is removed from solution by adsorption onto charcoal, and an aliquot of the supernatant fluid is counted by liquid scintillation techniques. The method is suitable for the assay of PRPP synthetase in crude hemolysates so long as NaF is added to inhibit ATPase activity, and each assay tube is corrected for cpm obtained in a control reaction from which ribose-5-phosphate is omitted. The assay mixture contained 50 rnxf triethanolamine-100 mh,r potassium phosphate buffer, pH 8.0 ( 19), 5 mM ribose-5-phosphate, 3 mM ATP-Y-“*P ( 1OO,OOO-200,000 cpm), 5 mM MgCl,, 25 MM NaF, and enzyme in a final volume of 0.50 ml. The reactions proceeded at 37” and were terminated by addition of 0.50 ml 5%perchloric acid. The reaction mixtures were placed on ice for 10-l-5 min and 0.3 ml 25% (v/v) acidwashed Norit (22) was added to adsorb unreacted ATP-y-““P. After lo15 min, 0.2 ml of “Carrie? (5 mg/ml bovine serum albumin in 50 mM sodium pyrophosphate, pH 7) was added to each tube to aggregate any fine particles of charcoal and ensure displacement of ““PPi from the charcoal. The tubes were centrifuged, and a OSO-ml aliquot of the supernatant liquid was added to 15 ml of scintillation fluid, which’consisted of 0.5% (w/v) diphenyloxazole in 8 parts methanol to 7 parts toltime by volume. Radioactivity was determined in a Beckman LS-100 scintilladon counter. The orotate removal assay for PRPP, which was used to establish the validity of the ATP-Y-~‘P removal assay, has been described (20). In our experience this assay responded linearly only in the range from 0.01 to 0.1 pmoles of PRPP per ml of reaction mixture. Protein was determined by the Folin method of Lowry et al. (23) using crystalline bovine serum albumin as a standard. Electrophoresis and Activity Stain for PRPP Sqnthetase. Electrophoresis
PRPP
SYNTHETASE
IN
EXCESSIVE
PURINE
PRODUCTION
269
was carried out at 4” on Cellogel cellulose acetate strips (H. ReeveAngel Co.), 2%10 cm wide and 17 cm long, in a Gellman electrophoresis apparatus. The Cellogel strips were equilibrated in the electrophoresis buffer (40 mM sodium barbital, 10 mM sodium phosphate, 10 mM glutathione, and 1 mM MgC12, final pH 8.6) for at least 10 min. Samples of 5-25 ~1 of partially purified PRPP synthetase preparation were applied with either an applicator or a micropipette, and the electrophoresis was begun at once. The electrophoresis was allowed to proceed from 30 to 60 min, usually 45 min, at 400 V and approximately 6 mA per each 2% cm of Cellogel strip width. Extending the time of electrophoresis beyond 1 hr generally resulted in loss of stainable activity. Immediately after the electrophoresis, the Cellogel strips were immersed in freshly prepared staining solution (described below) for 3-.5 min in the dark at room temperature. The intensity of staining was increased by incubating the strips for 15-20 min in a moist, dark chamber at 37”. The staining pattern was fixed and the strip cleared by soaking for 5-10 min in a solution of methanol-acetic acid-water (5: 1:.5). Each sample was compared to a control in which the staining solution did not contain PRPP. The activity stain is based upon the reverse reaction of PRPP synthetase:
TPN+
AMP
+ PRPP
--t
ATP
ATP
+ glucose
--f
glucose-6-P
-
TPNH
+ glucose-6-P
+ ribose-5-P
f
+ ADP 6-phosphogluconste
PMS
TPNH
+ nitrobluetetrazolium
-+
blue-black
stain.
The staining solution is made up of the following: 5 ml 0.1 M triethanolamine-O.1 M phosphate buffer, pH 8.0, 100 ,Lmole glucose, 50 pmole MgC12, 25 pmole AMP, 15 pmole PRPP, 10 pmole TPN+, 50 pmole creatine phosphate, 20 units glucose-6-phosphate dehydrogenase, 40 units hexokinase, 50 units creatine phosphokinase, 10 mg bovine serum albumin, 0.4 mg phenazine methosulfate (PMS ), and 4 mg nitrobluetetrazolium in a final volume of 9.4 ml. The staining solution must be made up immediately before use and is stable for only about 30 min. Creatine phosphate and creatine phosphokinase were added as an ATP regenerating system to increase the sensitivity of the staining solution, RESULTS
Assay for PRPP Synthetase in Hemolysates. The 3ZP transfer assay for PRPP synthetase (see Methods) proved to be suitable for determining the enzyme in freshly prepared hemolysates. Results of a typical assay
270
JOHNSON
0
5
ET
10 Time (minutes)
AL.
15
Xl
FIG. 1. Assay of PRPP synthetase in hemolysates using the “P transfer assay. The sample was from normal erythrocytes and was prepared and assayed as described in Materials and Methods. The protein concentration of the hemolysate was 57 mg/ml. (A) Linearity of assay with amount of hemolysate. (B) Linearity of assay with time. Each point is corrected for an identical control reaction mixture from which ribose-S-phosphate was omitted.
are shown in Fig. 1. The assay simply determines the release of 32P from ATP-Y-~~P into any form not adsorbable by charcoal; when controls lacking ribosed-phosphate are subtracted, 32P release not related to PRPP synthesis is eliminated. The validity of equating ribose-Sphosphatedependent release of 32P from ATP-Y-~~P to PRPP synthesis was demonstrated by an independent enzymic assay for PRPP. Reaction mixtures identical to one used for the 32P assay were used, except that the reactions were terminated by heating at 100” for 60 set and cooled on ice. Aliquots were then assayed for PRPP by enzymic conversion to orotidylic acid and uridylic acid, which was followed spectrophotometrically (20). After correction for a small amount of decomposition of PRPP during the heating used to terminate the reaction (7.5%), the pmoles of PRPP found by enzymic assay averaged 88% of the pmoles of 32P released from ATP in the 32P transfer assay. The small difference between the two assays is probably due to breakdown or metabolic
PRPP
SYNTHETASE
IN
EXCESSIVE
PUFlINE
271
PRODUCTION
utilization of a small amount of PRPP during the first step of the orotate assay. The PRPP synthetase of human erythrocytes is rather unstable. Washed erythrocytes could be frozen and stored at - 10” without significant loss of activity for up to 12 wk, although samples stored for longer periods appeared to progressively lose PRPP synthetase activity. Freezing and thawing of the erythrocytes brought about a 20% loss of the activity after each freeze-thaw cycle. The hemolysates were quite unstable to even a single freeze-thaw treatment. Thus, to obtain reliable assays of PRPP synthetase in human erythrocytes, the cells must be washed and stored as described in Methods and assayed after only a single thawing within a few weeks of collection. Observing these precautions, we have measured the PRPP synthetase activities in the erythrocytes of 23 normal individuals, ranging in age from 21 to 42 yr, and in a few patients with well-defined pathologies (Table 1) . We were able to demonstrate that the erythrocytes of LeschNyhan patients possessed normal PRPP synthetase activity. This finding confirms an earlier conclusion (7) that the elevated levels of PRPP in the tissue of Lesch-Nyhan patients do not result from excessive PRPP synthetase activity. A patient afflicted with Tay-Sachs disease also had normal PRPP synthetase activity, as would be expected. Of greatest interest, we have determined the PRPP synthetase activity in a sample of erythrocytes from a patient (T.B.), who has been shown to possess elevated PRPP synthetase activity associated with gout (12). As shown in Table 1, the activity determined was well above the normal range. TABLE PRPP
SYNTHETASE
Subject
Medical status
Normal volunteers 6 F, 17 M M.J. T.B.
-
J.C. C.B. $k..
1
ACTIWTIICS IN ERYTHROCYTE: LYSATES OF NORMAL AND INDIVIDUALLY WITH METABOLIC DISEASES
Lesch-Nyhan syndrome Elevated PRPP synthetase associated with gout* Gout Gout Tay-Sachs disease
Erythrocyte
SUBJECTS
PRPP synthetase activity
nmoles PRPP/min/mg Ave = 2.06 Range = 1.6&2.81 2.35, 2.44a 3.58
protein
2.08 2.16 1.93
a Duplicate determinations on different days and different hemolysates. * This patient has been described previously (12). The sample analyzed had been stored for 7 mo (see text).
272
JOHNSOK
ET
AL.
We believe, however, that the sample should actually show an appreciably higher value. The only sample available to LIS had been stored for 7 11.10; a sample collected at the same time from a normal relative of the patient (B.B., Ref. 12) yielded activity of 1.10 nmole/min/mg protein, which is only about half of the normal value obtained with fresh erythrocyte samples. An assay of the 7-mo-old samples with the methods used previously (12) confirmed that they had lost 3040% of their initial activity on storage. There is little doubt, therefore, that the PRPP synthetase mutation that has been detected by an entirely different assay method would also be readily detected by the :‘-P transfer method if reasonably fresh erythrocytes were assayed. of PRPP Synthetase. The localElectrophoresis and Activity Wining ization of PRPP synthetase activity on Cellogel strips after electrophoresis was accomplished by an activity stain, which is based on coupling of the reverse reaction of the enzyme to formation of TPNH, as described above. Because of the presence of contaminating activities in the partially purified hemolysates, it was necessary to compare the staining pattern obtained with and without PRPP in the staining solution (Fig. 2, left and right panels, respectively). It is not known whether the dark band that forms on such control strips is due to the so-called “nothing dehydrogenase” that has been reported by others (24, 25) or to another reaction that forms ATP from the staining components. Figure 2 shows an example of the electrophoretic activity-staining experiments. The PRPP synthetase from normal individuals migrate in a position closest to the origin of any staining activity, well behind a clear zone, which always appears and serves as a convenient marker on the electropherograms. We have examined partially purified preparations of PRPP
FIG. 2. Electropherogram of partially purified PRPP synthetase after development with the specific activity stain. Left panel: complete staining solution. Right panel: control in which staining solution lacked PRPP. T.B. is a patient with mutant PRPP synthetase (12) and is compared with a sample from a normal individual.
PRPP
SYNTHETASE
IN
EXCESSIVE
PURINE
PRODUCTION
273
synthetase from more than one dozen normal individuals; in all cases the electrophoretic mobility of the enzyme was identical and essentially the same as in the example shown in Fig. 2. The mobility of PRPP synthetase from patient T.B., who has been shown to possess elevated PRPP synthetase levels ( 12), is clearly different from the normal enzyme (Fig. 2). Note that the mutant enzyme migrates to the same position as the clear zone and is clearly separated from the normal enzyme. These experiments demonstrate the utility of the activity stain on cellulose acetate electrophoresis strips for detecting PRPP synthetase and the feasability of detecting mutant forms of PRPP synthetase by this technique. DISCUSSION
The activity of human PRPP synthetase has been assayed by a variety of techniques. The most common method involves a procedure in which the PRPP generated in the synthetase reaction is reacted with a radioactively labeled base in the presence of the appropriate partially purified purine phosphoribosyltransferase ( 21, 26, 27). Nonisotopic assays have also been described (28). The 32P transfer method described here is rapid, very sensitive, and if properly controlled, completely specific for PRPP synthetase activity. The assay does not require secondary incubations or chromatographic or electrophoretic separation of reaction products. The procedure does not require coupling enzymes and hence is free from artifacts that could result from unsuspected interferences with the activity of the coupling enzyme. Since neither PRPP, nor the products of PRPP hydrolysis, nor the products of subsequent PRPP metabolism (primarily PPi in each case) are adsorbed onto charcoal, the assay is not limited by the chemical and metabolic instability of PRPP. A possible disadvantage to some clinical laboratories of the :12P transfer assay is the requirement for a supply of “2P-ATP. However, we have found the procedure of Glynn and Chappell ( 18) for preparing 12PATP to be simple, efficient, and quite inexpensive. The hazards of working with the levels of ‘BP required for these assaysare very minor. The particular assay conditions described here for the 32Ptransfer assay call for high concentration of Pi and probably would not detect the type of PRPP synthetase anomaly reported by Sperling et UZ. ( 13, 14), in which the enzyme is hyperactive only at low concentrations of Pi. However, the assay can readily be modified by substitution of a low Pi concentration to detect such anomalies. Similarly, the assay is readily adaptable to the study of substrate saturation or inhibition by end products by steadystate kinetic techniques. The activity stain for PRPP synthetase reported here should be useful in screening for electrophoretic variants of PRPP synthetase and thereby
274
JOHNSON
ET
AL.
locating possible mutations in the enzyme that might not be detected by an activity assay under standard conditions. The activity stain has also proven useful in demonstrating that the alteration in PRPP synthetase activity exhibited by patient T.B. results from a structural change in the enzyme, rather than from a mutation that alters the amount of the normal enzyme (29). A disadvantage of the assay stain is the fact that we have been unable to apply it successfully to crude hemolysates, SO that a purification step is necessary. We have not made significant efforts toward applying the activity stain to extracts of other tissues; preliminary attempts to locate PRPP synthetase on electropherograms of crude extracts of fibroblasts have not been successful. SUMMARY
A simple and sensitive method for the assay of phosphoribosylpyrophosphate (PRPP) synthetase activity in lysates of human erythrocytes is described. The assay, which is based on the ribose S-phosphatedependent transfer of radioactivity from ATP-Y-~*P to PRPP, does not require coupling enzymes nor electrophoretic nor chromatographic separation of reaction products. A procedure for locating the activity of PRPP synthetase on cellulose acetate strips after electrophoresis is also presented. The detection technique relies on use of enzymes coupled to the reverse reaction of PRPP synthetase to generate a PRPP-dependent stain, Both procedures are shown to detect a previously described mutant form of PRPP synthetase and should be of use in the study of metabolic disorders that may involve alterations in PRPP synthetase activity. Note added in proof. The clear zone consistently seen on electropherograms in a position just ahead of normal PRPP synthetase activity (Fig. 2) is probably the result of superoxide dismutase activity, which is present in human erythrocytes and has been recently shown to prevent photochemical reduction of tetrazolium dyes [Lippitt, B., and Fridovich, I., Arch. Biochem. Biophys. 159, 739 (1973)]. ACKNOWLEDGMENTS The research was supported by Public Health Service Grant No. AM R.L.S. and Grants No. AM 13622, AM 05646, and GM 17702 to J.E.S.
13488
to
REFERENCES 1. SEEGMILLER, Rosenberg, 2.
WOOD,
3. CASKEY, 2570 4. HOLMES, 6035
J, E., Eds.),
in “Duncan’s Diseases of Metabolism” (P. K. Bondy and L. Ed. 6, p. 516. W. B. Saunders, Philadelphia, ( 1969). A. W., AND SEEGMILLER, J. E., J. Biol. Chem. 248, 138 ( 1973). C. T., ASHTON, D. M., AND WYNGAARDEN, J. B., 1. Biol. Chem. 239, ( 1964). E. W., WYNGAARDEN, J. B., AND KELLEY, W. N., I. Biol. Chem. 248, ( 1973 ) .
PRPP
5. LESCH, M., 6. SEEGMILLER,
SYNTHETASE
IN
AND NYHAN, W. L., J. E., ROSENBLQOM,
EXCESSIVE
PURINE
PRODUCHON
275
Amer. J. Med. 36, 561 (1964). F. M., AND KELLEY, W. N., Science 155, 1682
(1967). 7. ROSENBLOOM,
F. M., HENDERSON, J. F., CALDWELL, I. C., KELLEY, W. AND SEEGMILLER, J. E., J. Biol. Chem. 243,1166 (1968). 8. GREEN, M. L., AND SEECXILLER, J. E., Arthritis Rheum. 12, 666 (1969). 9. Fox, I. H., AND KELLEY, W. N., Ann. Intern. Med. 74, 424 (1971). 10. WOOD, A. W., BECKER, M. A., AND SEEGMILLER, J. E., Biochem. Genet. 9, (1973). 11. SPERLING, O., BOER, P., PERSKY-BROSH, S., KANAREK, E., AND DEVRIES,
N.,
261 A.,
Europ. J. Clin. Biol. Res. 17, 703 ( 197%). 12. BECKER, M. A., MEYER, L. J., WOOD, A. W., AND SEEGMILLER, J. E., Science 179, 1123 (1973). 13. SPERLING, O., EILAM, C., PERSKY-BROSH, S., AND DEVRIES, A., Biochem. Med. 6,310 (1972). 14. SPERLING, O., PERSKY-BROSH,
S., BOER,
P., AND DEVRIES,
A., Biochem. Med. 7,
389 ( 1973). 15. HERSHKO, A., HERSHKO,
C., AND MAGER, J., Israel 1. Med. Sci. 4, 939 ( 1968). F. L., AND WILLIAMS, H. C., Metabolism 20, 737 (1971). W. N., ANDERSON, H. M., PAGLIA, D. E., JAFFEE, E. R., KOSRAD, AND HARRIS, S. R., Blood 39, 674 ( 1972). I. M., AND CHAPPELL, J. B., Biochem. J. 90, 147 (1964).
16. MEYSKENS, 17. VALENTINE,
P. N., 18. CLYNN, 19. SWITZER, R. L., j. Biol. Chem. 216, 2447 (1971). 20. KORNUERG, A., LIEBERMAN, I., AND SIMMS, E. S., J. Biol. Chem. 215, 389 ( 1955). 21. Fox, I. H., AND KELLEY, W. N., J. Biol. Chem. 246, 5739 ( 1971). 22. ZIMMERMAN, S. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. VI, p. 258. Academic Press, New York, 1963. 23. LO-Y, 0. H., ROSEBROUGH, N. O., FARR, A. L., AND RANDALL, R. J., 1. Biol. Chem. 193,265 (1951). 24. HORI, S. H., AND KAWAMURA, T., Acta Histochem. Cytochem. I, 95 ( 1968). 25. ZIMMERMAN, H., AND PEARSE, A. G. E., 1. Histochem. Cytochem. 7, 271 (1959). 26. HENDERSON, J. F., AND KHOO, M. Y., J. Biol. Chem. 240, 2349 (1965). 27. HERSHKO, A., RAZIN, A., AND MAGER, J., Biochem. Biophys. Acta 184, 64 (1969). 28. VALENTINE, W. N., AND KURSCHNER, K. K., Blood 39, 666 ( 1972). 29. BECKER, M. A., KOSTEL, P. J., MEYER, L. J., AND SEEGMILLER, J. E., Proc. Nat. Acad. Sci. U.S.A. ‘70, 2749 (1973).
of
the
Role
phate Synthetase Gout: A Simple
of
Department
-Pyrophos-
in Congenital Hyperuricemia and Isotopic Assay and an Activity
Stain MARTIN
5-Phosphoribosyl-a-1
for the Enzyme
G. JOHNSON, AND ROBERT
of Bioc.hc.mistr!y.
SIMON ROSENZWEIG L. SWITZER
Vniversitl/
of lliinoi.c,
Urbana.
2llinoi.r
6!ROl
AND
MICHAEL Department
A. BECKER of Medicine,
University California Received
J. EDWIN
AND
of Californk 92037
October
SEEGMILLER at Sal& Diego,
La Jolla,
29, 1973
INTRODUCTION
Among the mechanisms that have been suggested for the excessive uric acid production of a significant proportion of patients with primnry metabolic gout are defects that lead to excessive concentrations of phosphoribosylpyrophosphate (PRPP) , a substrate for the first step of purine biosynthesis de no~o (1). This reaction, catalyzed by PRPP:glutamine amidotransferase, is sensitive to feedback inhibition by purine nucleotides; these nucleotides, while probably binding at specific allosteric sites, are kinetically competitive with respect to PRPP (24). Thus, an elevation of PRPP concentrations above normal levels would be expected to increase the rate of purine synthesis not only because of increased availability of a substrate for PRPP : glutamine amidotransferasc, but also as a result of a desensitization of the first enzyme of purine synthesis to feedback control, possibly through a conversion from the less active dimer to a more active monomeric form (4). The view that PRPP concentration may be an important element in excessive uric acid production in man first received experimental support from studies of the Lesch-Nyhan syndrome, an inherited disease characterized by mental retardation, choreoathetosis, self-mutilation, and extreme over-production of uric acid (5). The Lesch-Nyhan syndrome Copyright .4Il rights
266 @ 1974 by Academic Press, Inc. of reproduction in any forln rrserwd.
PRPP
SYNTHETASE
IN
EXCESSIVE
PURINE
PRODUCTION
267
results from a severe deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (6). Erythrocytes, fibroblasts, and lymphoblasts from affected individuals show substantially increased concentrations of the free PRPP (7-10). Subsequently, Sperling et ~2. (11) and Becker et al. (12) have independently reported the occurrence of families with inherited hyperuricemia and gout in which erythrocytes from affected individuals possess elevated PRPP synthetase (EC 2.7.6.1) activity. The fact that the abnormal PRPP synthetase activities discovered by these two groups have very different kinetic properties suggests that a variety of PRPP synthetase defects may be associated with purine overproduction ( 12-14). It is clear, however, that, in many hyperuricemic or gouty individuals with excessive purine production, altered levels of PRPP or of PRPP synthetase activity cannot be detected, at least in erythrocytes (8, 9, 15, 16). Recently, an instance of hemolytic anemia associated with a deficiency of PRPP synthetase has b-en described ( 17). The frequency of occurrence and variety of metabolic disorders resulting from abnormalities in the functioning of PRPP synthetase remains to be fully established. This paper presents a method for sensitive. specific, and relatively simple determination of PRPP synthetase activity in human erythrocyte lysates that is free of some of the problems of other techniques. In addition, an activity stain for PRPP synthetase that has proven useful in studying electrophoretic behavior of normal and mutant enzymes is presented. These methods should prove useful in diagnosis and study of metabolic disorders that may involve PRPP concentrations and PRPP synthetasc activity. MATERIALS
AND METHODS
Materials. Adenosine triphosphate, ribose-5-phosphate, creatine phosphate, TPN”, phenazine methosulfate, and nitrobluetetrazolium were purchased from Calbiochem or Sigma. PRPP, dimagnesium salt, was the product of P-L Biochemicals, Inc. ATP-y-“‘P was prepared by the exchange procedure of Glynn and Chappel ( la), purified, and characterized as previously described ( 19). Hexokinase, glucose-6-phosphate dehydrogenase, creatine phosphokinase, and the enzymes used in the synthesis of ATP-Y-“~P were highly purified preparations obtained from Sigma. The mixture of orotidylic pyrophosphorylase and orotidylic decarboxylase used for PRPP assays by the orotate removal method (20) was a crude preparation from yeast sold by P-L Biochemicals, Inc. Preparation of Hemolysates. Blood was collected by venipuncture into heparinized tubes, which were kept on ice. Erythrocytes were separated from the plasma by centrifugation at 1900 X g for 15 min and washed three times with 4 vol of cold 0.9%NaCI, buffered to pH 7.4 by addition
of 1 M tris-Cl, pIf 7.4. The packed cells were stored at - 10” until use. Hemolysates were prepared from frozen crythrocytes by thawing the cells in 3 vol of cold 1 rnM EDTA, pH 7. After standing at O-2” for 15 min, lysis of the erythrocytes was complete. Partial Purification of PRPP Synthetase for Activity Staining. The activity stain has not been successfully applied to crude hemolysates; hence, the PRPP synthetase used in activity staining was partially (5090-fold) purified from 10-15 ml of packed, washed cells using a slight modification of the DEAE-cellulose batch elution step previously described by Fox and Kelley (21). Th e volumes of solutions used we’-e all l/20 of the volumes listed, and intermediate washing of the DEAEcellulose with 3 mM sodium phosphate-50 rnM KC1 buffer was omitted since it did not improve the purification significantly. Assa!y for PRPP Synthetase. The assny for PRPP synthetase is based on enzymic transfer of radioactivity from ATP-y-“‘P to PRPP and/or its acid degradation products. The unreacted ATP is removed from solution by adsorption onto charcoal, and an aliquot of the supernatant fluid is counted by liquid scintillation techniques. The method is suitable for the assay of PRPP synthetase in crude hemolysates so long as NaF is added to inhibit ATPase activity, and each assay tube is corrected for cpm obtained in a control reaction from which ribose-5-phosphate is omitted. The assay mixture contained 50 rnxf triethanolamine-100 mh,r potassium phosphate buffer, pH 8.0 ( 19), 5 mM ribose-5-phosphate, 3 mM ATP-Y-“*P ( 1OO,OOO-200,000 cpm), 5 mM MgCl,, 25 MM NaF, and enzyme in a final volume of 0.50 ml. The reactions proceeded at 37” and were terminated by addition of 0.50 ml 5%perchloric acid. The reaction mixtures were placed on ice for 10-l-5 min and 0.3 ml 25% (v/v) acidwashed Norit (22) was added to adsorb unreacted ATP-y-““P. After lo15 min, 0.2 ml of “Carrie? (5 mg/ml bovine serum albumin in 50 mM sodium pyrophosphate, pH 7) was added to each tube to aggregate any fine particles of charcoal and ensure displacement of ““PPi from the charcoal. The tubes were centrifuged, and a OSO-ml aliquot of the supernatant liquid was added to 15 ml of scintillation fluid, which’consisted of 0.5% (w/v) diphenyloxazole in 8 parts methanol to 7 parts toltime by volume. Radioactivity was determined in a Beckman LS-100 scintilladon counter. The orotate removal assay for PRPP, which was used to establish the validity of the ATP-Y-~‘P removal assay, has been described (20). In our experience this assay responded linearly only in the range from 0.01 to 0.1 pmoles of PRPP per ml of reaction mixture. Protein was determined by the Folin method of Lowry et al. (23) using crystalline bovine serum albumin as a standard. Electrophoresis and Activity Stain for PRPP Sqnthetase. Electrophoresis
PRPP
SYNTHETASE
IN
EXCESSIVE
PURINE
PRODUCTION
269
was carried out at 4” on Cellogel cellulose acetate strips (H. ReeveAngel Co.), 2%10 cm wide and 17 cm long, in a Gellman electrophoresis apparatus. The Cellogel strips were equilibrated in the electrophoresis buffer (40 mM sodium barbital, 10 mM sodium phosphate, 10 mM glutathione, and 1 mM MgC12, final pH 8.6) for at least 10 min. Samples of 5-25 ~1 of partially purified PRPP synthetase preparation were applied with either an applicator or a micropipette, and the electrophoresis was begun at once. The electrophoresis was allowed to proceed from 30 to 60 min, usually 45 min, at 400 V and approximately 6 mA per each 2% cm of Cellogel strip width. Extending the time of electrophoresis beyond 1 hr generally resulted in loss of stainable activity. Immediately after the electrophoresis, the Cellogel strips were immersed in freshly prepared staining solution (described below) for 3-.5 min in the dark at room temperature. The intensity of staining was increased by incubating the strips for 15-20 min in a moist, dark chamber at 37”. The staining pattern was fixed and the strip cleared by soaking for 5-10 min in a solution of methanol-acetic acid-water (5: 1:.5). Each sample was compared to a control in which the staining solution did not contain PRPP. The activity stain is based upon the reverse reaction of PRPP synthetase:
TPN+
AMP
+ PRPP
--t
ATP
ATP
+ glucose
--f
glucose-6-P
-
TPNH
+ glucose-6-P
+ ribose-5-P
f
+ ADP 6-phosphogluconste
PMS
TPNH
+ nitrobluetetrazolium
-+
blue-black
stain.
The staining solution is made up of the following: 5 ml 0.1 M triethanolamine-O.1 M phosphate buffer, pH 8.0, 100 ,Lmole glucose, 50 pmole MgC12, 25 pmole AMP, 15 pmole PRPP, 10 pmole TPN+, 50 pmole creatine phosphate, 20 units glucose-6-phosphate dehydrogenase, 40 units hexokinase, 50 units creatine phosphokinase, 10 mg bovine serum albumin, 0.4 mg phenazine methosulfate (PMS ), and 4 mg nitrobluetetrazolium in a final volume of 9.4 ml. The staining solution must be made up immediately before use and is stable for only about 30 min. Creatine phosphate and creatine phosphokinase were added as an ATP regenerating system to increase the sensitivity of the staining solution, RESULTS
Assay for PRPP Synthetase in Hemolysates. The 3ZP transfer assay for PRPP synthetase (see Methods) proved to be suitable for determining the enzyme in freshly prepared hemolysates. Results of a typical assay
270
JOHNSON
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5
ET
10 Time (minutes)
AL.
15
Xl
FIG. 1. Assay of PRPP synthetase in hemolysates using the “P transfer assay. The sample was from normal erythrocytes and was prepared and assayed as described in Materials and Methods. The protein concentration of the hemolysate was 57 mg/ml. (A) Linearity of assay with amount of hemolysate. (B) Linearity of assay with time. Each point is corrected for an identical control reaction mixture from which ribose-S-phosphate was omitted.
are shown in Fig. 1. The assay simply determines the release of 32P from ATP-Y-~~P into any form not adsorbable by charcoal; when controls lacking ribosed-phosphate are subtracted, 32P release not related to PRPP synthesis is eliminated. The validity of equating ribose-Sphosphatedependent release of 32P from ATP-Y-~~P to PRPP synthesis was demonstrated by an independent enzymic assay for PRPP. Reaction mixtures identical to one used for the 32P assay were used, except that the reactions were terminated by heating at 100” for 60 set and cooled on ice. Aliquots were then assayed for PRPP by enzymic conversion to orotidylic acid and uridylic acid, which was followed spectrophotometrically (20). After correction for a small amount of decomposition of PRPP during the heating used to terminate the reaction (7.5%), the pmoles of PRPP found by enzymic assay averaged 88% of the pmoles of 32P released from ATP in the 32P transfer assay. The small difference between the two assays is probably due to breakdown or metabolic
PRPP
SYNTHETASE
IN
EXCESSIVE
PUFlINE
271
PRODUCTION
utilization of a small amount of PRPP during the first step of the orotate assay. The PRPP synthetase of human erythrocytes is rather unstable. Washed erythrocytes could be frozen and stored at - 10” without significant loss of activity for up to 12 wk, although samples stored for longer periods appeared to progressively lose PRPP synthetase activity. Freezing and thawing of the erythrocytes brought about a 20% loss of the activity after each freeze-thaw cycle. The hemolysates were quite unstable to even a single freeze-thaw treatment. Thus, to obtain reliable assays of PRPP synthetase in human erythrocytes, the cells must be washed and stored as described in Methods and assayed after only a single thawing within a few weeks of collection. Observing these precautions, we have measured the PRPP synthetase activities in the erythrocytes of 23 normal individuals, ranging in age from 21 to 42 yr, and in a few patients with well-defined pathologies (Table 1) . We were able to demonstrate that the erythrocytes of LeschNyhan patients possessed normal PRPP synthetase activity. This finding confirms an earlier conclusion (7) that the elevated levels of PRPP in the tissue of Lesch-Nyhan patients do not result from excessive PRPP synthetase activity. A patient afflicted with Tay-Sachs disease also had normal PRPP synthetase activity, as would be expected. Of greatest interest, we have determined the PRPP synthetase activity in a sample of erythrocytes from a patient (T.B.), who has been shown to possess elevated PRPP synthetase activity associated with gout (12). As shown in Table 1, the activity determined was well above the normal range. TABLE PRPP
SYNTHETASE
Subject
Medical status
Normal volunteers 6 F, 17 M M.J. T.B.
-
J.C. C.B. $k..
1
ACTIWTIICS IN ERYTHROCYTE: LYSATES OF NORMAL AND INDIVIDUALLY WITH METABOLIC DISEASES
Lesch-Nyhan syndrome Elevated PRPP synthetase associated with gout* Gout Gout Tay-Sachs disease
Erythrocyte
SUBJECTS
PRPP synthetase activity
nmoles PRPP/min/mg Ave = 2.06 Range = 1.6&2.81 2.35, 2.44a 3.58
protein
2.08 2.16 1.93
a Duplicate determinations on different days and different hemolysates. * This patient has been described previously (12). The sample analyzed had been stored for 7 mo (see text).
272
JOHNSOK
ET
AL.
We believe, however, that the sample should actually show an appreciably higher value. The only sample available to LIS had been stored for 7 11.10; a sample collected at the same time from a normal relative of the patient (B.B., Ref. 12) yielded activity of 1.10 nmole/min/mg protein, which is only about half of the normal value obtained with fresh erythrocyte samples. An assay of the 7-mo-old samples with the methods used previously (12) confirmed that they had lost 3040% of their initial activity on storage. There is little doubt, therefore, that the PRPP synthetase mutation that has been detected by an entirely different assay method would also be readily detected by the :‘-P transfer method if reasonably fresh erythrocytes were assayed. of PRPP Synthetase. The localElectrophoresis and Activity Wining ization of PRPP synthetase activity on Cellogel strips after electrophoresis was accomplished by an activity stain, which is based on coupling of the reverse reaction of the enzyme to formation of TPNH, as described above. Because of the presence of contaminating activities in the partially purified hemolysates, it was necessary to compare the staining pattern obtained with and without PRPP in the staining solution (Fig. 2, left and right panels, respectively). It is not known whether the dark band that forms on such control strips is due to the so-called “nothing dehydrogenase” that has been reported by others (24, 25) or to another reaction that forms ATP from the staining components. Figure 2 shows an example of the electrophoretic activity-staining experiments. The PRPP synthetase from normal individuals migrate in a position closest to the origin of any staining activity, well behind a clear zone, which always appears and serves as a convenient marker on the electropherograms. We have examined partially purified preparations of PRPP
FIG. 2. Electropherogram of partially purified PRPP synthetase after development with the specific activity stain. Left panel: complete staining solution. Right panel: control in which staining solution lacked PRPP. T.B. is a patient with mutant PRPP synthetase (12) and is compared with a sample from a normal individual.
PRPP
SYNTHETASE
IN
EXCESSIVE
PURINE
PRODUCTION
273
synthetase from more than one dozen normal individuals; in all cases the electrophoretic mobility of the enzyme was identical and essentially the same as in the example shown in Fig. 2. The mobility of PRPP synthetase from patient T.B., who has been shown to possess elevated PRPP synthetase levels ( 12), is clearly different from the normal enzyme (Fig. 2). Note that the mutant enzyme migrates to the same position as the clear zone and is clearly separated from the normal enzyme. These experiments demonstrate the utility of the activity stain on cellulose acetate electrophoresis strips for detecting PRPP synthetase and the feasability of detecting mutant forms of PRPP synthetase by this technique. DISCUSSION
The activity of human PRPP synthetase has been assayed by a variety of techniques. The most common method involves a procedure in which the PRPP generated in the synthetase reaction is reacted with a radioactively labeled base in the presence of the appropriate partially purified purine phosphoribosyltransferase ( 21, 26, 27). Nonisotopic assays have also been described (28). The 32P transfer method described here is rapid, very sensitive, and if properly controlled, completely specific for PRPP synthetase activity. The assay does not require secondary incubations or chromatographic or electrophoretic separation of reaction products. The procedure does not require coupling enzymes and hence is free from artifacts that could result from unsuspected interferences with the activity of the coupling enzyme. Since neither PRPP, nor the products of PRPP hydrolysis, nor the products of subsequent PRPP metabolism (primarily PPi in each case) are adsorbed onto charcoal, the assay is not limited by the chemical and metabolic instability of PRPP. A possible disadvantage to some clinical laboratories of the :12P transfer assay is the requirement for a supply of “2P-ATP. However, we have found the procedure of Glynn and Chappell ( 18) for preparing 12PATP to be simple, efficient, and quite inexpensive. The hazards of working with the levels of ‘BP required for these assaysare very minor. The particular assay conditions described here for the 32Ptransfer assay call for high concentration of Pi and probably would not detect the type of PRPP synthetase anomaly reported by Sperling et UZ. ( 13, 14), in which the enzyme is hyperactive only at low concentrations of Pi. However, the assay can readily be modified by substitution of a low Pi concentration to detect such anomalies. Similarly, the assay is readily adaptable to the study of substrate saturation or inhibition by end products by steadystate kinetic techniques. The activity stain for PRPP synthetase reported here should be useful in screening for electrophoretic variants of PRPP synthetase and thereby
274
JOHNSON
ET
AL.
locating possible mutations in the enzyme that might not be detected by an activity assay under standard conditions. The activity stain has also proven useful in demonstrating that the alteration in PRPP synthetase activity exhibited by patient T.B. results from a structural change in the enzyme, rather than from a mutation that alters the amount of the normal enzyme (29). A disadvantage of the assay stain is the fact that we have been unable to apply it successfully to crude hemolysates, SO that a purification step is necessary. We have not made significant efforts toward applying the activity stain to extracts of other tissues; preliminary attempts to locate PRPP synthetase on electropherograms of crude extracts of fibroblasts have not been successful. SUMMARY
A simple and sensitive method for the assay of phosphoribosylpyrophosphate (PRPP) synthetase activity in lysates of human erythrocytes is described. The assay, which is based on the ribose S-phosphatedependent transfer of radioactivity from ATP-Y-~*P to PRPP, does not require coupling enzymes nor electrophoretic nor chromatographic separation of reaction products. A procedure for locating the activity of PRPP synthetase on cellulose acetate strips after electrophoresis is also presented. The detection technique relies on use of enzymes coupled to the reverse reaction of PRPP synthetase to generate a PRPP-dependent stain, Both procedures are shown to detect a previously described mutant form of PRPP synthetase and should be of use in the study of metabolic disorders that may involve alterations in PRPP synthetase activity. Note added in proof. The clear zone consistently seen on electropherograms in a position just ahead of normal PRPP synthetase activity (Fig. 2) is probably the result of superoxide dismutase activity, which is present in human erythrocytes and has been recently shown to prevent photochemical reduction of tetrazolium dyes [Lippitt, B., and Fridovich, I., Arch. Biochem. Biophys. 159, 739 (1973)]. ACKNOWLEDGMENTS The research was supported by Public Health Service Grant No. AM R.L.S. and Grants No. AM 13622, AM 05646, and GM 17702 to J.E.S.
13488
to
REFERENCES 1. SEEGMILLER, Rosenberg, 2.
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3. CASKEY, 2570 4. HOLMES, 6035
J, E., Eds.),
in “Duncan’s Diseases of Metabolism” (P. K. Bondy and L. Ed. 6, p. 516. W. B. Saunders, Philadelphia, ( 1969). A. W., AND SEEGMILLER, J. E., J. Biol. Chem. 248, 138 ( 1973). C. T., ASHTON, D. M., AND WYNGAARDEN, J. B., 1. Biol. Chem. 239, ( 1964). E. W., WYNGAARDEN, J. B., AND KELLEY, W. N., I. Biol. Chem. 248, ( 1973 ) .
PRPP
5. LESCH, M., 6. SEEGMILLER,
SYNTHETASE
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AND NYHAN, W. L., J. E., ROSENBLQOM,
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F. M., HENDERSON, J. F., CALDWELL, I. C., KELLEY, W. AND SEEGMILLER, J. E., J. Biol. Chem. 243,1166 (1968). 8. GREEN, M. L., AND SEECXILLER, J. E., Arthritis Rheum. 12, 666 (1969). 9. Fox, I. H., AND KELLEY, W. N., Ann. Intern. Med. 74, 424 (1971). 10. WOOD, A. W., BECKER, M. A., AND SEEGMILLER, J. E., Biochem. Genet. 9, (1973). 11. SPERLING, O., BOER, P., PERSKY-BROSH, S., KANAREK, E., AND DEVRIES,
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C., AND MAGER, J., Israel 1. Med. Sci. 4, 939 ( 1968). F. L., AND WILLIAMS, H. C., Metabolism 20, 737 (1971). W. N., ANDERSON, H. M., PAGLIA, D. E., JAFFEE, E. R., KOSRAD, AND HARRIS, S. R., Blood 39, 674 ( 1972). I. M., AND CHAPPELL, J. B., Biochem. J. 90, 147 (1964).
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P. N., 18. CLYNN, 19. SWITZER, R. L., j. Biol. Chem. 216, 2447 (1971). 20. KORNUERG, A., LIEBERMAN, I., AND SIMMS, E. S., J. Biol. Chem. 215, 389 ( 1955). 21. Fox, I. H., AND KELLEY, W. N., J. Biol. Chem. 246, 5739 ( 1971). 22. ZIMMERMAN, S. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. VI, p. 258. Academic Press, New York, 1963. 23. LO-Y, 0. H., ROSEBROUGH, N. O., FARR, A. L., AND RANDALL, R. J., 1. Biol. Chem. 193,265 (1951). 24. HORI, S. H., AND KAWAMURA, T., Acta Histochem. Cytochem. I, 95 ( 1968). 25. ZIMMERMAN, H., AND PEARSE, A. G. E., 1. Histochem. Cytochem. 7, 271 (1959). 26. HENDERSON, J. F., AND KHOO, M. Y., J. Biol. Chem. 240, 2349 (1965). 27. HERSHKO, A., RAZIN, A., AND MAGER, J., Biochem. Biophys. Acta 184, 64 (1969). 28. VALENTINE, W. N., AND KURSCHNER, K. K., Blood 39, 666 ( 1972). 29. BECKER, M. A., KOSTEL, P. J., MEYER, L. J., AND SEEGMILLER, J. E., Proc. Nat. Acad. Sci. U.S.A. ‘70, 2749 (1973).