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Altered isoelectric property of a superactive 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase in a patient with clinical gout
BIOCHEMICAL
MEDICINE
26,
387-394 (1981)
Altered Isoelectric Property of a Superactive 5Phosphoribosyl-1 -pyrophosphate (PRPP) Synthetase in a Patient with Clinical Gout YUTARO Department
NISHIDA,
IEO AKAOKA,
AND YOSHIHIKO
HORIUCHI
of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo ll3? Japan
Received March 10, 1981
5-Phosphoribosyl-I-pyrophosphate (PRPP) is a high-energy compound which is synthesized from ribose 5-phosphate and ATP by the action of PRPP synthetase. Its intracellular concentration has been emphasized as an important factor in determining the rate of de nova purine biosynthesis (1). Furthermore, Sperling et al. (2,3), de Vries and Sperling (4), Zoref et al. (5), and Becker et al. (6-10) have demonstrated that some superactive variants of PRPP synthetase were present in erythrocytes and fibroblasts obtained from four cases; that the variants led to de ~OVOpurine overproduction; and that the variants differed from normal PRPP synthetase in kinetics and physical properties. Thus, they suggested that the variants might result from structural gene mutation. We have measured PRPP synthetase activity in erythrocyte lysates from 110patients with clinical gout. The activity of the enzyme obtained from one of these patients was proved to be about twice higher than the mean value. The present study was designed to elucidate the physical and chemical properties of PRPP synthetase in this patient. MATERIALS
AND METHODS
The materials used in the present study were [8-14C]adenine (62 mCi/ mmole) and [8-14C]hypoxathine (59 mCi/mmole) (Radiochemical Centre); disodium salt PRPP (P-L Biochemicals, Inc.); uricase (Seikagaku Kogyo Corp.); ribose S-phosphate, ATP, 2,3-diphosphoglycerate (2,3-DPG), adenosine 5-monophosphate (AMP), and other nonradioactive purines (Sigma Chemical Co.). Blood samples were collected from 110gouty subjects by venepuncture into heparinized tubes. Erythrocytes were separated from the plasma by 387 0006-2944/81/060387-08$02.0010 Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any fern, reserved.
388
NISHIDA,
AKAOKA,
AND
HORIUCHI
centrifugation at 19OOg at 4°C for 15 min. After washing twice with 4 vol of cold physiological saline, 0.5 ml of the packed washed erythrocytes was hemolyzed in 3.0 ml of 1 mM EDTA. Erythrocyte lysates were then dialyzed at 4°C for 12 hr against 8 mM sodium phosphate buffer (pH 7.4) containing 1 mM EDTA and 5 mM reduced glutathion. PRPP synthetase activity in dialyzed erythrocyte lysates was measured by a minor modification of the two-step method of Becker et al. (6). Dialyzed erythrocyte lysates (25 ~1) were added to 0.5 ml of the firststep reaction mixture which contained 0.5 mM ATP, 0.35 mM ribose 5phosphate, 1 mM EDTA, 25 mM MgCL 2.5 mM reduced glutathione, 32 mM sodium phosphate buffer (pH 7.4), and 50 mM Tris-HCl (pH 7.4). This reaction mixture was incubated for 20 min at 37°C. The reaction was terminated by the addition of 100 )~.l of cold 100 mM EDTA. The tubes were heated for 1 min in a boiling water bath, and then cooled in an ice-water bath. Two hundred fifty microliters of 10% charcoal solution was added to each tube. This mixture was shaken vigorously. After centrifugation at 2000g for 10 mitt, 100 ~1 of the supernate was assayed for PRPP content. In the second step of the assay, [14C]adenine was converted to [14C]AMP by PRPP in the presence of highly purified APRTase. One hundred microliters of the heat-treated supernate was added to 100 t.~l of 100 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgC12, 202 FM [14C]adenine, and 10 cl.1of APRTase. After incubation for 30 min at 37”C, 25 ~1 of the reaction mixture was spotted on Eastman Kodak thin-layer chromatography sheets. The sheets were developed with butanol, methanol, water, and 25% NH,OH (60:20:20:1 v/v) until the solvent front moved up to 15 cm. Then the sheets were air-dried. The purines were located on the chromatogram under ultraviolet light. Adenylic acid spots were cut out and suspended in Aquasol II solution (New England Nuclear). 14C was counted in an Aloca liquid scintillation counter. Erythrocyte PRPP concentration was determined by the method of Becker et al. (6). Freshly drawn and packed red cells (25 ~1) were added to 275 ~1 of 50 mM Tris-HCl buffer (pH 7.4) containing 1 mr+r EDTA. The tubes were heated for 1 min in a boiling water bath and then cooled in an ice-water bath. After centrifugation at 19OOg for 10 min at 5°C 100 ~1 of the supernate was assayed for PRPP content as described above. APRTase was purified from human erythrocytes by the method of Thomas et al. (11). Erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) activity was assayed after the dialyzed hemolysate was incubated with 2 mM PRPP and 800 PM [‘4C]hypoxanthine in 100 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl, (12). The formed [‘4C]inosinic acid was separated and measured by the same method as used for the determination of the PRPP concentration. Plasma
5-PHOSPHORIBOSYL-I-PYROPHOSPHATE
SYNTHETASE
389
and urinary urate levels were measured by the enzymatic method of Liddle el al. (13). Protein concentration was determined by the method of Lowry et al. (14). Furthermore, the properties of PRPP synthetase in a patient and a healthy subject were investigated using dialyzed erythrocyte lysates. PRPP-synthetase activity was determined at varying concentrations of inorganic phosphate (Pi) from 1 .O to 32 mM and at the fixed concentrations of ATP and ribose Sphosphate. Km values were determined by standard Lineweaver-Burk plots obtained from reaction mixtures containing the fixed concentration of ATP and the varying concentrations of ribose 5phosphate and those containing the fixed concentration of ribose Sphosphate and the varying concentrations of ATP. The inhibition of PRPP synthetase was studied at the fixed levels of ATP and ribose 5-phosphate and the varying concentrations of ADP, AMP, and 2,3-DPG. Thermal inactivation of erythrocyte lysates in the patient and control was also studied. Equal volumes of dialyzed erythrocyte lysates were incubated at various time intervals at 60°C. The lysates were put in an ice-water bath and used to determine PRPP synthetase activity. To further characterize the enzyme properties, electrofocusing was performed using the technique previously described by Haglund (15). One milliliter of erythrocyte hemolysates was applied to the LKB ampholine electrofocusing column. A pH linear gradient of 200 ml of a mixture containing 1 to 40% sucrose and 1% ampholytes (pH 3 to 10) was formed at 4°C for 48 hr. Five milliliters of each fraction was collected. The pH of every fraction was measured with a Horiba pH meter. The fractions were also assayed for PRPP synthetase activity. CASE REPORT A 28-year-old male patient experienced severe pain and swelling of the metatarsophalangeal joint of the left foot for 1 week. One of his cousins on his father’s side was affected with gout. Physical examinations revealed normal findings except mild obesity (body weight of 91 kg and height of 153.4 cm) and blood pressure of 172/110 mm Hg. There was no tophus. Routine urinalysis was negative. Laboratory tests revealed serum uric acid of 9.2 mg/lOO ml and 24-hr urinary urate excretion of 885 mg on a put-me-free diet. Other laboratory data were within normal range. The bone X ray was read as normal. RESULTS The mean erythrocyte PRPP synthetase activity in the 110 gouty patients included in the present study was 47.8 ? 11.1 nmole/mg protein/ hr. However, the activity of this enzyme in one of the patients was 92 nmole/mg protein/hr-about twice higher than the mean value. The eryth-
390
NISHIDA,
AKAOKA,
AND HORIUCHI
rocyte PRPP concentration in this patient was 12.4 nmole/ml packed cells, which was markedly higher than those in other gouty patients. Other characteristics of erythrocyte PRPP synthetase obtained from this patient are described below in comparison with the enzyme obtained from a normal subject. Changes in erythrocyte PRPP synthetase activity at the varying concentrations of Pi and the fixed concentrations of ATP and ribose 5phosphate are shown in Fig. 1. The sigmoidal response of PRPP synthetase activity to the increasing concentrations of Pi was observed in both the patient and the normal subject. The patient’s PRPP synthetase activity was about twice higher than the normal value in the entire range of Pi concentrations. The K, values of erythrocyte PRPP synthetase to ATP and ribose 5phosphate in the patient were 6.25 and 14.9 FM, respectively, which were identical with the normal values. The patient’s erythrocyte PRPP synthetase activity was inhibited by 0.1 mM AMP, 0.5 mM ADP, and 10 mM 2,3-DPG to 60, 55, and 30% of the corresponding control values, respectively. However, there was no difference in sensitivity to these inhibitors between the enzyme of the patient and that of the normal subject. The heat stability of erythrocyte PRPP synthetase is shown in Fig. 2. After 10 min of incubation of the erythrocyte lysates at 60°C the patient’s enzyme became unstable and its activity decreased to 30% of the preincubation value, while the activity of the normal enzyme decreased to 50% of the preincubation value.
Concentration
of Pi (mM)
1. PRPP synthetase activity in dialyzed hemolysates at the varying concentrations of inorganic phosphate (Pi) and the fixed concentrations of ATP and ribose Sphosphate. FIG.
S-PHOSPHORIBOSYL-I-PYROPHOSPHATE
patient
0
3
5
J. T .
15
10 Time
391
SYNTHETASE
(minutes)
FIG. 2. Heat stability of erythrocyte PRPP synthetase. Erythrocyte lysates were incubated for the indicated time intervals at 60°C. PRPP synthetase was assayed after the lysates were transferred to the ice-water bath.
The elution profile of isoelectric focusing of erythrocyte PRPP synthetase is shown in Fig. 3. Normal PRPP synthetase still showed only one peak at pH 5.02 after it had been aggregated by addition of 150 mM KCl. On the other hand, the patient’s PRPP synthetase showed two peaks (peak I at pH 4.3 and peak II at pH 5.011, being less stable to heat at peak I than at peak II.
k-t
8 -
5 b
6 .
O-
s 5
IO
20 Fraction
30
40
number
FIG. 3. Elution profile of isoelectric focusing of PRPP synthetase in erythrocytes from the patient and the normal subject. The gradually rising line shows pH. Each fraction was assayed for PRPP synthetase activity. Patient (0); Normal subject (0).
392
NISHIDA,
AKAOKA,
AND
HORIUCHI
DISCUSSION Molecular abnormalities in PRPP synthetase have been demonstrated by Sperling et al. (3), de Vries and Sperling (4), and Becker et al. (6,lO). We also found a superactive form of this enzyme in erythrocytes obtained from one of the 110 gouty patients included in this study. Its activity was about twice higher than the mean value. The physical and chemical properties of erythrocyte PRPP synthetase in this patient were compared with those of the enzyme in a normal subject and the previous findings of other investigators. Although Sperling et al. (3) reported that the Pi activation curve of the mutant form of PRPP synthetase took a hyperbolic shape, the curve of the enzyme obtained from our patient and normal subject showed a sigmoidal pattern. However, the degree of Pi activation of the enzyme within the range of Pi concentrations from 1 to 32 mM was always about two times higher in the patient than in the normal subject. The present study demonstrated that little difference was found in K,,, values for ATP and ribose 5-phosphate between the normal and superactive forms of erythrocyte PRPP synthetase. Similar findings were obtained by Sperling et al. (3) and Becker et al. (6). The present study also showed that although the patient’s erythrocyte PRPP synthetase was inhibited by AMP, ADP, and 2,3-DPG, no difference was found in the degree of inhibition by these substances between the normal and superactive forms of the enzyme. A similar finding was obtained by Becker et al. (6). On the other hand, Sperling et al. (3) observed that the sensitivity of the mutant form of the enzyme to these inhibitors was diminished. The superactive form of erythrocyte PRPP synthetase obtained from the present study became less stable at 60°C than the normal form. However, Sperling et al. (3) and Becker et al. (6) reported that little difference was found in thermal lability between the normal and superactive forms of the enzyme. Becker et al. (16) purified erythrocyte PRPP synthetase and performed electrophoresis using polyacrylamide gel. They detected an electrophoretie mobility distinct from that of the normal form of PRPP synthetase. This method was not employed in the present study because a limited quantity of blood sample from the patient prevented the purification of the enzyme. Isoelectric focusing was performed to further elucidate the mutant properties of erythrocyte PRPP synthetase obtained from our patient. Becker ef al. (16) and Fox and Kelley (17) demonstrated only one isoelectric point in the normal and superactive forms of the enzyme. Interestingly, the present study showed the appearance of two peaks in
5-PHOSPHORIBOSYL-I-PYROPHOSPHATE
393
SYNTHETASE
ihe patient’s enzyme: peak I at pH 4.3 and peak II at pH 5.01. To check whether one of these two peaks was attributable to aggregation, the normal form of the enzyme was treated with KC1 based on the finding of Becker et al. (18) that PRPP synthetase was aggregated by 150 mM KCl. However, the normal form of the enzyme was proved to show only one isoelectric focusing point at pH 5.02 before and after KC1 treatment. This suggested that the isoelectric focusing point of peak I observed in the present study might not be attributable to aggregation, and that the enzyme at peak I might have a different molecular form resulting from gene alteration. Zoref et al. (19), Yen et al. (20), and Becker et al. (21) have reported that the gene of human PRPP synthetase was mapped on the X chromosome. No blood relationship was demonstrated between the mother of our patient and the mother of his gouty cousin. Gout in the cousin is therefore thought to have been caused by another mechanism, although his PRPP synthetase activity was not measured. Gene alteration may have occurred in the patient himself or on the female line of his mother. SUMMARY
Erythrocyte Sphosphoribosyl-I-pyrophosphate (PRPP) synthetase activity was assayed to screen possible variants of this enzyme in 110 patients with clinical gout. The enzyme obtained from one of these patients proved to have the following characteristics: (a) Its activity was about twice higher than the mean value. (b) Its activity was sigmoidally elevated by inorganic phosphate (Pi) over a wide range of Pi concentrations from 1 to 32 mM. (c) Its K, values to ATP and ribose 5-phosphate were identical with the control values. (d) The degree to which it was inhibited by AMP, ADP, and 2,3-DPG did not differ from the finding obtained from the control. (e) It was very unstable at 60°C. (D Isoelectric focusing revealed that it had an elution profile with two peaks (peak I at pH 4.3 and peak II at pH 5.01, being less stable to heat at peak I than at peak II. These findings suggested that erythrocyte PRPP synthetase obtained from the patient might be a variant resulting from the alteration of its molecular form. REFERENCES 1,
2. 3. 4. 5. 6.
Fox, I. H., and Kelley, W. N., Ann. Intern. Med. 74, 424 Sperling, O., Eilam, G., Persky-Brosh, S., and de Vries, (1972). Sperling, O., Persky-Brosh, S., Boer, P.. and de Vries, (1973). de Vries, A., and Sperling, O., Urologic A12, 153 (1973). Zoref, E., de Vries, A., and Sperling, 0.. 1. Cfin. fnvest. Becker, M. A., Meyer, L. J., and Seegmiller, J. E., Amer.
(1971).
A., Biochem. Med.
6, 310
A., Biochem.
7, 389
Med.
56, 1093 (1975). J. Med. 55, 232 (1973).
394
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AKAOKA,
AND HORIUCHI
7. Becker, M. A., Kostel, P. J., Meyer, L. J., and Seegmiller, J. E., Proc. Nat. Acad. Sri. USA 70, 2749 (1973). 8. Becker, M. A., Kostel, P. J., and Meyer, L. J., J. Biol. Chem. 250, 6822 (1975). 9. Becker, M. A., J. Clin. Invest. 57, 308 (1976). 10. Becker, M. A., Raivio, K. O., Bakay, B., Adams, W. B., and Nyhan. W. L., J. Ch. Invest. 65, 109 (1980). 11. Thomas, C. B., Arnold, W. J., and Kelley, W. N., J. Biol. Chem. 248, 2529 (1973). 12. Kelley, W. N., Rosenbloom, F. M., Henderson, J. A., and Seegmiller, J. E., Proc. Nat. Acad. Sci. USA 57, 1735 (1967). 13. Liddle, L., Seegmiller, J. E., and Laster, L., J. Lab. Clin. Med. 54, 901-903 (1959). 14. Lowry, 0. H., Rosebrough. N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). 15. Haglund, H. Methods Biochem. Anal. 19, 1 (1971). 16. Becker, M. A., Kostel, P. J., and Meyer, L. J., J. Biol. Chem. 250, 6822 (1975). 17. Fox, I. H., and Kelley, W. N., J. Biol. Chem. 246, 5739 (1971). 18. Becker, M. A., Meyer, L. J., Huisman, W. H.. Lazar, C.. and Adams, W. B.. J. Biol. Chem. 252, 3911 (1977). 19. Zoref, E., de Vries, A., and Sperling, O., Advan. Exp. Med. Biol. 76A. 287 (1977). 20. Yen, R. C. K., Adams, W. B., Lazar, C.. and Becker, M. A., Proc. Nat. Acad. Sci. USA 75, 482 (1978). 21. Becker, M. A., Yen, R. C. K., Itkin, P.. Goss, S. J., Seegmiller, J. E., and Bakay, B., Science 203, 1016 (1979).
MEDICINE
26,
387-394 (1981)
Altered Isoelectric Property of a Superactive 5Phosphoribosyl-1 -pyrophosphate (PRPP) Synthetase in a Patient with Clinical Gout YUTARO Department
NISHIDA,
IEO AKAOKA,
AND YOSHIHIKO
HORIUCHI
of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo ll3? Japan
Received March 10, 1981
5-Phosphoribosyl-I-pyrophosphate (PRPP) is a high-energy compound which is synthesized from ribose 5-phosphate and ATP by the action of PRPP synthetase. Its intracellular concentration has been emphasized as an important factor in determining the rate of de nova purine biosynthesis (1). Furthermore, Sperling et al. (2,3), de Vries and Sperling (4), Zoref et al. (5), and Becker et al. (6-10) have demonstrated that some superactive variants of PRPP synthetase were present in erythrocytes and fibroblasts obtained from four cases; that the variants led to de ~OVOpurine overproduction; and that the variants differed from normal PRPP synthetase in kinetics and physical properties. Thus, they suggested that the variants might result from structural gene mutation. We have measured PRPP synthetase activity in erythrocyte lysates from 110patients with clinical gout. The activity of the enzyme obtained from one of these patients was proved to be about twice higher than the mean value. The present study was designed to elucidate the physical and chemical properties of PRPP synthetase in this patient. MATERIALS
AND METHODS
The materials used in the present study were [8-14C]adenine (62 mCi/ mmole) and [8-14C]hypoxathine (59 mCi/mmole) (Radiochemical Centre); disodium salt PRPP (P-L Biochemicals, Inc.); uricase (Seikagaku Kogyo Corp.); ribose S-phosphate, ATP, 2,3-diphosphoglycerate (2,3-DPG), adenosine 5-monophosphate (AMP), and other nonradioactive purines (Sigma Chemical Co.). Blood samples were collected from 110gouty subjects by venepuncture into heparinized tubes. Erythrocytes were separated from the plasma by 387 0006-2944/81/060387-08$02.0010 Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any fern, reserved.
388
NISHIDA,
AKAOKA,
AND
HORIUCHI
centrifugation at 19OOg at 4°C for 15 min. After washing twice with 4 vol of cold physiological saline, 0.5 ml of the packed washed erythrocytes was hemolyzed in 3.0 ml of 1 mM EDTA. Erythrocyte lysates were then dialyzed at 4°C for 12 hr against 8 mM sodium phosphate buffer (pH 7.4) containing 1 mM EDTA and 5 mM reduced glutathion. PRPP synthetase activity in dialyzed erythrocyte lysates was measured by a minor modification of the two-step method of Becker et al. (6). Dialyzed erythrocyte lysates (25 ~1) were added to 0.5 ml of the firststep reaction mixture which contained 0.5 mM ATP, 0.35 mM ribose 5phosphate, 1 mM EDTA, 25 mM MgCL 2.5 mM reduced glutathione, 32 mM sodium phosphate buffer (pH 7.4), and 50 mM Tris-HCl (pH 7.4). This reaction mixture was incubated for 20 min at 37°C. The reaction was terminated by the addition of 100 )~.l of cold 100 mM EDTA. The tubes were heated for 1 min in a boiling water bath, and then cooled in an ice-water bath. Two hundred fifty microliters of 10% charcoal solution was added to each tube. This mixture was shaken vigorously. After centrifugation at 2000g for 10 mitt, 100 ~1 of the supernate was assayed for PRPP content. In the second step of the assay, [14C]adenine was converted to [14C]AMP by PRPP in the presence of highly purified APRTase. One hundred microliters of the heat-treated supernate was added to 100 t.~l of 100 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgC12, 202 FM [14C]adenine, and 10 cl.1of APRTase. After incubation for 30 min at 37”C, 25 ~1 of the reaction mixture was spotted on Eastman Kodak thin-layer chromatography sheets. The sheets were developed with butanol, methanol, water, and 25% NH,OH (60:20:20:1 v/v) until the solvent front moved up to 15 cm. Then the sheets were air-dried. The purines were located on the chromatogram under ultraviolet light. Adenylic acid spots were cut out and suspended in Aquasol II solution (New England Nuclear). 14C was counted in an Aloca liquid scintillation counter. Erythrocyte PRPP concentration was determined by the method of Becker et al. (6). Freshly drawn and packed red cells (25 ~1) were added to 275 ~1 of 50 mM Tris-HCl buffer (pH 7.4) containing 1 mr+r EDTA. The tubes were heated for 1 min in a boiling water bath and then cooled in an ice-water bath. After centrifugation at 19OOg for 10 min at 5°C 100 ~1 of the supernate was assayed for PRPP content as described above. APRTase was purified from human erythrocytes by the method of Thomas et al. (11). Erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) activity was assayed after the dialyzed hemolysate was incubated with 2 mM PRPP and 800 PM [‘4C]hypoxanthine in 100 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl, (12). The formed [‘4C]inosinic acid was separated and measured by the same method as used for the determination of the PRPP concentration. Plasma
5-PHOSPHORIBOSYL-I-PYROPHOSPHATE
SYNTHETASE
389
and urinary urate levels were measured by the enzymatic method of Liddle el al. (13). Protein concentration was determined by the method of Lowry et al. (14). Furthermore, the properties of PRPP synthetase in a patient and a healthy subject were investigated using dialyzed erythrocyte lysates. PRPP-synthetase activity was determined at varying concentrations of inorganic phosphate (Pi) from 1 .O to 32 mM and at the fixed concentrations of ATP and ribose Sphosphate. Km values were determined by standard Lineweaver-Burk plots obtained from reaction mixtures containing the fixed concentration of ATP and the varying concentrations of ribose 5phosphate and those containing the fixed concentration of ribose Sphosphate and the varying concentrations of ATP. The inhibition of PRPP synthetase was studied at the fixed levels of ATP and ribose 5-phosphate and the varying concentrations of ADP, AMP, and 2,3-DPG. Thermal inactivation of erythrocyte lysates in the patient and control was also studied. Equal volumes of dialyzed erythrocyte lysates were incubated at various time intervals at 60°C. The lysates were put in an ice-water bath and used to determine PRPP synthetase activity. To further characterize the enzyme properties, electrofocusing was performed using the technique previously described by Haglund (15). One milliliter of erythrocyte hemolysates was applied to the LKB ampholine electrofocusing column. A pH linear gradient of 200 ml of a mixture containing 1 to 40% sucrose and 1% ampholytes (pH 3 to 10) was formed at 4°C for 48 hr. Five milliliters of each fraction was collected. The pH of every fraction was measured with a Horiba pH meter. The fractions were also assayed for PRPP synthetase activity. CASE REPORT A 28-year-old male patient experienced severe pain and swelling of the metatarsophalangeal joint of the left foot for 1 week. One of his cousins on his father’s side was affected with gout. Physical examinations revealed normal findings except mild obesity (body weight of 91 kg and height of 153.4 cm) and blood pressure of 172/110 mm Hg. There was no tophus. Routine urinalysis was negative. Laboratory tests revealed serum uric acid of 9.2 mg/lOO ml and 24-hr urinary urate excretion of 885 mg on a put-me-free diet. Other laboratory data were within normal range. The bone X ray was read as normal. RESULTS The mean erythrocyte PRPP synthetase activity in the 110 gouty patients included in the present study was 47.8 ? 11.1 nmole/mg protein/ hr. However, the activity of this enzyme in one of the patients was 92 nmole/mg protein/hr-about twice higher than the mean value. The eryth-
390
NISHIDA,
AKAOKA,
AND HORIUCHI
rocyte PRPP concentration in this patient was 12.4 nmole/ml packed cells, which was markedly higher than those in other gouty patients. Other characteristics of erythrocyte PRPP synthetase obtained from this patient are described below in comparison with the enzyme obtained from a normal subject. Changes in erythrocyte PRPP synthetase activity at the varying concentrations of Pi and the fixed concentrations of ATP and ribose 5phosphate are shown in Fig. 1. The sigmoidal response of PRPP synthetase activity to the increasing concentrations of Pi was observed in both the patient and the normal subject. The patient’s PRPP synthetase activity was about twice higher than the normal value in the entire range of Pi concentrations. The K, values of erythrocyte PRPP synthetase to ATP and ribose 5phosphate in the patient were 6.25 and 14.9 FM, respectively, which were identical with the normal values. The patient’s erythrocyte PRPP synthetase activity was inhibited by 0.1 mM AMP, 0.5 mM ADP, and 10 mM 2,3-DPG to 60, 55, and 30% of the corresponding control values, respectively. However, there was no difference in sensitivity to these inhibitors between the enzyme of the patient and that of the normal subject. The heat stability of erythrocyte PRPP synthetase is shown in Fig. 2. After 10 min of incubation of the erythrocyte lysates at 60°C the patient’s enzyme became unstable and its activity decreased to 30% of the preincubation value, while the activity of the normal enzyme decreased to 50% of the preincubation value.
Concentration
of Pi (mM)
1. PRPP synthetase activity in dialyzed hemolysates at the varying concentrations of inorganic phosphate (Pi) and the fixed concentrations of ATP and ribose Sphosphate. FIG.
S-PHOSPHORIBOSYL-I-PYROPHOSPHATE
patient
0
3
5
J. T .
15
10 Time
391
SYNTHETASE
(minutes)
FIG. 2. Heat stability of erythrocyte PRPP synthetase. Erythrocyte lysates were incubated for the indicated time intervals at 60°C. PRPP synthetase was assayed after the lysates were transferred to the ice-water bath.
The elution profile of isoelectric focusing of erythrocyte PRPP synthetase is shown in Fig. 3. Normal PRPP synthetase still showed only one peak at pH 5.02 after it had been aggregated by addition of 150 mM KCl. On the other hand, the patient’s PRPP synthetase showed two peaks (peak I at pH 4.3 and peak II at pH 5.011, being less stable to heat at peak I than at peak II.
k-t
8 -
5 b
6 .
O-
s 5
IO
20 Fraction
30
40
number
FIG. 3. Elution profile of isoelectric focusing of PRPP synthetase in erythrocytes from the patient and the normal subject. The gradually rising line shows pH. Each fraction was assayed for PRPP synthetase activity. Patient (0); Normal subject (0).
392
NISHIDA,
AKAOKA,
AND
HORIUCHI
DISCUSSION Molecular abnormalities in PRPP synthetase have been demonstrated by Sperling et al. (3), de Vries and Sperling (4), and Becker et al. (6,lO). We also found a superactive form of this enzyme in erythrocytes obtained from one of the 110 gouty patients included in this study. Its activity was about twice higher than the mean value. The physical and chemical properties of erythrocyte PRPP synthetase in this patient were compared with those of the enzyme in a normal subject and the previous findings of other investigators. Although Sperling et al. (3) reported that the Pi activation curve of the mutant form of PRPP synthetase took a hyperbolic shape, the curve of the enzyme obtained from our patient and normal subject showed a sigmoidal pattern. However, the degree of Pi activation of the enzyme within the range of Pi concentrations from 1 to 32 mM was always about two times higher in the patient than in the normal subject. The present study demonstrated that little difference was found in K,,, values for ATP and ribose 5-phosphate between the normal and superactive forms of erythrocyte PRPP synthetase. Similar findings were obtained by Sperling et al. (3) and Becker et al. (6). The present study also showed that although the patient’s erythrocyte PRPP synthetase was inhibited by AMP, ADP, and 2,3-DPG, no difference was found in the degree of inhibition by these substances between the normal and superactive forms of the enzyme. A similar finding was obtained by Becker et al. (6). On the other hand, Sperling et al. (3) observed that the sensitivity of the mutant form of the enzyme to these inhibitors was diminished. The superactive form of erythrocyte PRPP synthetase obtained from the present study became less stable at 60°C than the normal form. However, Sperling et al. (3) and Becker et al. (6) reported that little difference was found in thermal lability between the normal and superactive forms of the enzyme. Becker et al. (16) purified erythrocyte PRPP synthetase and performed electrophoresis using polyacrylamide gel. They detected an electrophoretie mobility distinct from that of the normal form of PRPP synthetase. This method was not employed in the present study because a limited quantity of blood sample from the patient prevented the purification of the enzyme. Isoelectric focusing was performed to further elucidate the mutant properties of erythrocyte PRPP synthetase obtained from our patient. Becker ef al. (16) and Fox and Kelley (17) demonstrated only one isoelectric point in the normal and superactive forms of the enzyme. Interestingly, the present study showed the appearance of two peaks in
5-PHOSPHORIBOSYL-I-PYROPHOSPHATE
393
SYNTHETASE
ihe patient’s enzyme: peak I at pH 4.3 and peak II at pH 5.01. To check whether one of these two peaks was attributable to aggregation, the normal form of the enzyme was treated with KC1 based on the finding of Becker et al. (18) that PRPP synthetase was aggregated by 150 mM KCl. However, the normal form of the enzyme was proved to show only one isoelectric focusing point at pH 5.02 before and after KC1 treatment. This suggested that the isoelectric focusing point of peak I observed in the present study might not be attributable to aggregation, and that the enzyme at peak I might have a different molecular form resulting from gene alteration. Zoref et al. (19), Yen et al. (20), and Becker et al. (21) have reported that the gene of human PRPP synthetase was mapped on the X chromosome. No blood relationship was demonstrated between the mother of our patient and the mother of his gouty cousin. Gout in the cousin is therefore thought to have been caused by another mechanism, although his PRPP synthetase activity was not measured. Gene alteration may have occurred in the patient himself or on the female line of his mother. SUMMARY
Erythrocyte Sphosphoribosyl-I-pyrophosphate (PRPP) synthetase activity was assayed to screen possible variants of this enzyme in 110 patients with clinical gout. The enzyme obtained from one of these patients proved to have the following characteristics: (a) Its activity was about twice higher than the mean value. (b) Its activity was sigmoidally elevated by inorganic phosphate (Pi) over a wide range of Pi concentrations from 1 to 32 mM. (c) Its K, values to ATP and ribose 5-phosphate were identical with the control values. (d) The degree to which it was inhibited by AMP, ADP, and 2,3-DPG did not differ from the finding obtained from the control. (e) It was very unstable at 60°C. (D Isoelectric focusing revealed that it had an elution profile with two peaks (peak I at pH 4.3 and peak II at pH 5.01, being less stable to heat at peak I than at peak II. These findings suggested that erythrocyte PRPP synthetase obtained from the patient might be a variant resulting from the alteration of its molecular form. REFERENCES 1,
2. 3. 4. 5. 6.
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