Clinica Chimica Actu, 203 (1991) 143-152
143
0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
CCA 05128
Phosphoribosylpyrophosphate synthetase in human erythrocytes: assay and kinetic studies using high-performance liquid chromatography Ryozo Sakuma ‘, Toshihiro Nishina ‘, Hisashi Yamanaka 2, Naoyuki Kamatani 2, Kusuki Nishioka 2, Masako Maeda 3 and Akio Tsuji 3 ’ Department of Clinical Chemistry, Toranomon Hospital, and Okinaka Memorial Institute for Medical Research, Minato-ku, Tokyo, 2 Institute of Rheumatology, Tokyo Women’s Medical College, Shinjuku-ku, Tokyo, and ’ School of Pharmaceutical Sciences, Showu University, Shinagawa-ku, Tokyo (Japan) (Received 30 January 1991; revision received 17 August 1991; accepted 19 August 1991) Key words: High-performance
liquid chromatography; Phosphoribosylpyrophosphate Pyrimidine 5’-nucleotidase deficiency
synthetase; Gout;
Summary A method using high-performance liquid chromatography (HPLC) for determination of phosphoribosylpyrophosphate (PRPP) synthetase activity in human erythrocytes has been developed and PRPP synthetase activity on purine and pyrimidine metabolic disorders has been studied. Kinetic properties of erythrocyte PRPP synthetase of patients with gout and of a patient with pyrimidine 5’nucleotidase deficiency were compared with those of healthy subjects. The mean of PRPP synthetase activity of gouty patients was a little higher (P < 0.01) than that of healthy subjects. The response of the enzyme for ATP of gouty patients was different from that of healthy subjects. The shapes of activation curve of the enzyme for inorganic phosphate were hyperbolic in gouty patients and in a patient with pyrimidine 5’-nucleotidase deficiency.
Introduction Phosphoribosylpyrophosphate synthetase (PRPP synthetase, EC 2.7.6.1) is a purine de novo enzyme. PRPP synthetase catalyzes the synthesis of PRPP from
Correspondence to: R. Sakuma, Department of Clinical Laboratory, Toranomon Branch Hospital, 1-3-1 Kajigaya, Takatsu-ku, Kawasaki 213, Japan.
144
ribose-j-phosphate (R5P) and adenosine5’-triphosphate (ATP) in the presence of inorganic phosphate and magnesium. The clinical importance of PRPP synthetase has been increased by the discovery of the inherited superactivity of the enzyme in red blood cells from patients with gout and overproduction of uric acid [1,2]. The methods for assay of PRPP synthetase in red blood cells have been studied by several workers in recent years [3-81. Many of the current methods for PRPP synthetase assay are radiochemical techniques [3-51. Such radiochemical techniques have been difficult to apply in clinical laboratories, due to the needs for special equipment for handling radioactive substances and for several complicated procedures. Spectrophotometric methods using enzyme-linked system are convenient, but these methods are hampered by poor sensitivity or poor precision [6-81. A method using high-performance liquid chromatography (HPLC) does not need special equipment or complicated procedures. We have already developed a nonradiochemical method using HPLC for assay of purine salvage enzymes, and have obtained good sensitivity and good precision [9]. We have therefore developed a method using HPLC for determination of PRPP synthetase activity in human erythrocytes, and have applied the method to study of PRPP synthetase activity on purine and pyrimidine metabolic disorders: gout, adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) deficiency, xanthine oxidase (X0, EC 1.2.3.2) deficiency, and pyrimidine 5’-nucleotidase (P5N, EC 3.1.3.5) deficiency. The mean of PRPP synthetase activity in erythrocytes from patients with gout was found to be the same level as that of healthy subjects [5,10], but an increase in the enzyme activity of patients with gout has been reported by Hardwell et al. [ll]. A decrease in the activity of PRPP synthetase in erythrocytes from patients with P5N deficiency has been reported by Valentine et al. [12]. However, the nature of the decreased PRPP synthetase activity in PSN-deficient erythrocytes remains unclear. Thus, we have studied kinetic properties of PRPP synthetase of gouty patients and a patient with P5N deficiency. Materials and methods Principle
PRPP synthetase catalyzes the following reaction: R5P + ATP + PRPP + AMP. Adenosine-5’-monophosphate (AMP) produced by the reaction is converted into adenosine-5’-diphosphate (ADP) with adenylate kinase (AK, EC 2.7.4.3): AMP + ATP -+ 2 ADP. The rate of the reaction catalyzed by PRPP synthetase is followed by measuring the increase in absorbance of ADP after separation of ADP from AMP and ATP by a reversed-phase HPLC.
145 Reagents
AMP, ADP, ATP (sodium salt), AK, R5P (sodium salt), and reduced glutathione were purchased from Boehringer Mannheim GmbH (Mannheim, FRG). Charcoal was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dextran (T-70) was from Pharmacia Fine Chemicals (Uppsala, Sweden). All other reagents were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sample preparation
Venous blood samples, obtained from 27 healthy subjects, 28 gouty patients, nine patients with APRT deficiency, a patient with X0 deficiency, and a patient with P5N deficiency, were collected in heparinised tubes. After centrifugation at 650 X g for 5 min at 4°C the plasma and buffy coat were removed. The remaining red cells were washed three times with 154 mmol/l NaCl, and packed by centrifugation at 650 X g for 10 min at 4°C. The packed cells were frozen and stored at -80’ C without loss of enzyme activity. The packed red cells were lysed with freeze-thawing ( - 80 and 37°C) twice and diluted with 5 volume of a charcoal-dextran suspension containing 10 g/l charcoal and 1 g/l dextran in cold distilled water. After mixing for about 10 s in a vortex mixer, the lysates were left for 10 min at 4°C and then centrifuged at 10,000 x g for 10 min at 4°C. The supernatant were used as enzyme samples. Hemoglobin (Hb) was determined by the cyanmethemoglobin method [13]. Enzyme assay
A reaction mixture contained 40 mmol/l sodium phosphate buffer, pH 7.4, 1.0 mmol/l R5P, 1.4 mmol/l ATP, 6 mmol/l MgCl,, 1 mmol/l reduced glutathione, and 1.8 IU/ml AK in a final volume of 2.0 ml. The reaction mixture was incubated for 5 min at 37°C. ATP was prepared fresh immediately before the enzyme assay. The enzyme reaction was started by the addition of 0.1 ml of the red cell lysate. After further incubation at 37 ’ C, 0.5 ml of the reaction mixtures were withdrawn at 10 min and 30 min into a tube containing 0.5 ml of 0.6 mol/l perchloric acid. After mixing for about 10 s, the mixtures were left for 5 min and centrifuged at 2,000 x g for 5 min. The supernatant, neutralized by adding the same volume of 0.4 mol/l Na,HPO,, were used as samples for HPLC analysis. Apparatus and chromatographic
conditions
The HPLC apparatus (from Tosoh, Co. Ltd., Tokyo, Japan) consisted of a Model 803D pump, an AS-48 autosampler (sample loop, 50 ~1) and a Model UV 8 II spectrophotometer connected to a Model CP-8000 chromatoprocessor. The spectrophotometer was set at 260 nm and 0.04 absorbance units full scale. AI1 separations were performed at ambient temperature on a column (25 cm x 4.6 mm i.d.1 packed with Tosoh ODS-120A gel (5 pm particle size) and a guard filter
146
(Tosoh line filter G). The mobile phase was sodium phosphate buffer (40 mmol/l, pH 5.6) containing 10 ml of methanol/l, It was continually degassed by a Erma Model ERC-3510 degasser (Erma Optical Works Ltd., Tokyo, Japan) connected to the 803D pump; the flow rate was 0.8 ml/min at 75 kg/cm*. ADP was determined by comparing the peak area in the chromatogram with values obtained from a standard curve. PRPP synthetase activity was calculated from the increase of ADP for 20 min. Results Chromatograms
of reaction mixtures
Figure 1 shows the chromatograms of the reaction mixtures after the enzyme reaction of erythrocyte PRPP synthetase from a healthy subject. The retention times after injection for ATP, ADP, and AMP were 7.5, 10, and 21 min, respectively. The peak of ADP was clearly separated from ATP and no peaks of unknown components, which would interfere with ADP peak, were observed with the progress of the enzyme reaction. Precision and sensitivity
The within-run precision of the method (n = 10) was evaluated by testing hemolysate of a healthy subject. Mean +_SD was 1.16 + 0.031 pmol/min per g Hb and the coefficient of variation (CV) was 2.7%. The product (ADP) concentration increased linearly with the reaction time for 40 min. ADP concentration in the reaction mixture after the enzyme reaction for 20 min was 60 pmol/l. The
(a>
(b)
ATP
DP
-f 0
--+ 10 Tir
20 ( inin
)
0
i
?I.-
10 Time
20 ( min
)
Fig. 1. HPLC profiles of the reaction mixtures after the enzyme reaction of erythrocyte PRPP synthetase from a healthy subject. (a) is the reaction mixture incubated for 10 min, and (b) is for 30 min. Elution conditions are described in the text.
147
sensitivity (detection limit) of the method evaluated by 2 SD of blank reaction was 0.005 pmol/min per g Hb. Comparison of a spectrophotometric method
This method was compared with the spectrophotometric method of Valentine et al. [6]. The within-run precision of the spectrophotometric method (n = 10) was evaluated by testing the same sample used in the HPLC method. Mean k SD was 1.03 + 0.053 pmol/min per g Hb and the CV was 5.1%. 2 SD of blank reaction was 0.07 pmol/min per g Hb. The activity of PRPP synthetase in erythrocytes from 38 outpatients was measured with both methods. Mean + SD obtained by the HPLC method was 1.04 + 0.09 pmol/min per g Hb and mean f SD obtained by the spectrophotometric method was 0.91 f 0.16 pmol/min per g Hb. PRPP sjrzthetase activities
The activity of PRPP synthetase obtained with erythrocytes from 27 healthy subjects (12 males and 15 females) was 1.10 + 0.11 pmol/min per g Hb (mean + SD). The present method was applied to assay of PRPP synthetase in erythrocytes from 28 patients with gout with normal uric acid excretion, nine patients with APRT deficiency, a patient with X0 deficiency, and a patient with P5N deficiency. The results are shown in Table I. The mean of PRPP synthetase activity of 28 patients with gout was a little higher (P < 0.01, t test) than that of 27 healthy subjects. The activities of PRPP synthetase of nine patients with APRT deficiency were a little higher than the mean of healthy subjects. The activity of the patient with xanthine oxidase deficiency was the lower limit of normal range. The activity of the patient with P5N deficiency was 66% of the mean of healthy subjects.
TABLE I PRPP synthetase activities in erythrocytes from patients with purine metabolic disorders and a patient with P5N deficiency Activity &mol/min
Diagnosis
per g Iib)
Mean + SD Normal (n = 27) Gout (n = 28) APRT deficiency a Homozygote (QO/QO) Heterozygote (QO/l) (n = 3) Japanese-type (J/J) (n = 5) X0 deficiency PSN deficiency ’ Letters in parentheses
1.10+0.11 1.28kO.11 1.30 1.13kO.02 1.16+0.12 0.96 0.73
indicate the genotypes of APRT deficiency [141.
148
, g
(a>
,
(b)
1.5
z .E c 1.0 2 2 .2‘ 0.5 .>
10
,
20
30
40
0
(b)
2
4
6
6
P G-mrol/l> % (mnol/l> Fig. 3. The relationships between the concentrations of activators, inorganic phosphate (a) and magnesium (b), and the activities of PRPP synthetase of a healthy subject (01, a gouty patient co), and the patient with P5N deficiency (A ).The enzyme reaction of (a) was carried out in 0.1 mol/l Tris-HC1 buffer, pH 7.4, containing 1.0 mmol/l RSP, 1.4 mmol/l ATP, 6 mmol/l MgCI,, 1 mmol/l reduced glutathione, 1.8 W/ml AK, and various concentrations of inorganic phosphate.
149
PSN-deficient patient were compared with those of the healthy subject (Fig. 3). The shapes of activation curves for inorganic phosphate were hyperbolic. Discussion The radiochemical methods for PRPP synthetase assay are based on the detection of isotopically labelled products separated from the substrates with high-voltage electrophoresis or thin-layer chromatography [3-51. The radiochemical techniques have required special equipment and several complicated procedures. Our method does not need special equipment and complicated procedures. AMP produced by the enzyme reaction is metabolized with other enzymes in erythrocytes, so AMP was converted into ADP with AK in our method. The treatment of the lysate with charcoal-dextran has been incorporated in the assay procedure to remove nucleotides in erythrocytes. Termination of the enzyme reaction and deproteinization of the solution after the enzyme reaction were simultaneously carried out with perchloric acid [El. Perchloric acid was immediately neutralized with sodium phosphate to avoid the hydrolysis of ATP in perchloric acid solution. A spectrophotometric method using the enzyme-linked system was reported by Valentine et al. [6], and modifications of the assay procedure of Valentine et al. were reported [7,81. These methods are based on measuring the decrease in absorbance of NADH, and require monitoring for 60 min at 340 nm. In addition, these methods require highly diluting sample to avoid the increase in initial absorbance of reaction solution. The sensitivity and precision of the methods are poor: 2 SD of brank reaction was 8% of normal PRPP synthetase activity [S] and the within-run CVs were 3-7% [7,8]. Our method is based on measuring the increase in absorbance of ADP at 260 nm. The molar extinction coefficient of ADP at 260 nm is greater than that of NADH at 340 nm, and the dilution of sample in our method is less than that in the spectrophotometric methods. In our method, 2 SD of brank reaction was 0.005 pmol/min per g Hb corresponding to 0.5% of normal value and the within-run CV was 2.7%. The data are good as compared with the results obtained by the spectrophotometric method of Valentine et al. [6]: 2 SD of brank reaction was 0.07 Fmol/min per g Hb and the within-run CV was 5.1%. The value of 1.04 f 0.09 pmol/min per g Hb obtained by our method is similar to the value of the spectrophotometric method (0.91 k 0.16 pmol/min per g Hb). The normal range of PRPP synthetase activity in erythrocytes obtained by our method was 1.10 k 0.11 pmol/min per g Hb. The value is similar to previously reported values: 0.97 + 0.27, 1.08 _t 0.30, and 1.50 f 0.25 pmol/min per g protein by radiochemical methods [4,5,101, 0.57 -t 0.07 and 1.60 f 0.52 pmol/min per g protein by spectrophotometric methods [7,8]. In our method, the mean of PRPP synthetase activity of gouty patients with normal excretion of uric acid was a little higher (P < 0.01) than that of healthy subjects (Table I). The result is different from the data previously reported [5,10]. Becker et al. [5] reported that the mean of PRPP synthetase activity of normal subjects was 1.08 + 0.30 pmol/min per g protein and the mean of gouty patients
1.50
with normal production of uric acid was 1.15 f 0.28 pmol/min per g protein. Losman et al. [lo] also reported that the activity of normal subjects (1.50 + 0.25 pmol/min per g protein) was the same level as gouty patients with normal uric acid excretion (1.45 + 0.23 pmol/min per g protein). However, Hardwell et al. [ll] have found out a significant increase 0’ < 0.05) of the activity of PRPP synthetase in gouty patients (2.03 f 0.74 pmol/min per g protein) compared with the values for control subjects (1.60 f 0.52 pmol/min per g protein). The differences of these results could be contributed to the concentration of ATP rather than R5P using as substrate, since the response of PRPP synthetase for ATP in the gouty patient was different from that of the healthy subject (Fig. 2b). PRPP synthetase of the gouty patient gave higher activity than that of the healthy subject at 1.33 mmol/l ATP used in our method and in the method of Hardwell et al. [ll], but gave the same activity level as the healthy subject at 0.5 mmol/l ATP used in the methods of Becker et al. [5] and of Losman et al. [lo]. The same results as shown in Fig. 2b were obtained in other gouty patients and other healthy subjects (data not shown). The apparent K, value for R5P of the gouty patient was similar to that of the healthy subject, but the increase of V,,, was observed (Fig. 3aI. The shape of activation curve of PRPP synthetase for inorganic phosphate was hyperbolic in the gouty patient (Fig. 3a). The hyperbolic activation for inorganic phosphate has been found in normal hemolysates chromatographed on Sephadex G-25 [161 or treated with activated charcoal [31, while the sigmoidal activation has appeared in crude [3] or dialyzed hemolysates [lo]. The shape of activation curve for magnesium of the gouty patient was similar to that of the healthy subject (Fig. 3b). A decrease in the activity of PRPP synthetase in erythrocytes of hereditary P5N deficiency has been reported by Valentine et al. [12]. Paglia et al. [17] reported the increase of the K, value for R5P of PRPP synthetase in PSN-deficient erythrocytes, about two- and three-fold of normal. In this study, the marked increase of K, value for R5P was not obtained in the patient with P5N deficiency. The differences of these results could be related to the treatment of the lysate with charcoal-dextran to remove nucleotides. Fox and Kelley [181 observed that 1 mmol/l cytidine di- and triphosphate inhibited PRPP synthetase activity at nonsaturating substrate concentrations. The accumulated pyrimidine nucleotides, which inhibit PRPP synthetase activity, were removed from the PSN-deficient erythrocyte by charcoal in our method, so the increase of the K, value for R5P of PRPP synthetase was not observed in the PSN-deficient erythrocyte. Although the accumulated pyrimidine nucleotides were removed by charcoal and the enzyme reaction was performed in the presence of saturating substrate concentrations, the decreased PRPP synthetase activity was obtained in the PSN-deficient erythrocyte. The decreased PRPP synthetase activity could not be caused by changes of the response of the enzyme for activators, inorganic phosphate and magnesium, because the shapes of activation curves for inorganic phosphate and magnesium of the PSN-deficient patient were similar to those of the healthy subject. The state of the subunit aggregation of PRPP synthetase is relation with the enzyme activity [19,201. The molecular size of PRPP synthetase of the PSN-deficient erythrocyte was compared with that of a healthy subject by gel filtration. The
151
result obtained by gel filtration was similar to the data reported by Lachant et al. [Zll (data not shown). The decreased PRPP synthetase activity of the PSN-deficient erythrocyte could be related to the amount of aggregated forms rather than the amount of disaggregated forms. However, further studies are needed to clarify the nature of the decreased PRPP synthetase activity in PSN-deficient erythrocytes. Acknowledgement The authors wish to thank Dr. Shiro Miwa for providing a blood sample of the patient with pyrimidine 5’nucleotidase deficiency. References 1 Becker MA, Meyer LJ, Wood AW, Seegmiller JE. Purine overproduction in man associated with increased phosphoribosylpyrophosphate synthetase activity. Science 1973;179:1123-1126, 2 Sperling 0, Eilam S, Persky-Brosh S, de Vries A. Accelerated erythrocyte 5-phosphoribosyl-l-pyrophosphate synthesis. A familial abnormality associated with excessive uric acid production and gout. Biochem Med 1972;6:310-316. 3 Hershko A, Razin A, Mager J. Regulation of the synthesis of 5-phosphoribosyl-I-pyrophosphate in intact red blood cells and in cell-free preparations. Biochim Biophys Acta 1969;184:64-76. 4 Fox IH, Kelley WN. Human phosphoribosylpyrophosphate synthetase. J Biol Chem 1971;246:57395748. 5 Becker MA, Meyer LJ, Wood AW, Seegmiller JE. Gout with purine overproduction due to increased phosphori~sylpyrophosphate synthetase activity. Am J Med 1973;55:232-242. 6 Valentine WN, Kurschner KK. Studies on human erythrocyte nucleotide metabolism. I Nonisotopic methodologies. Blood 197239666-673. 7 Ferrari M, Giacomello A, Salerno C, Messina E. A spectrophotometric assay for phosphoribosyl pyrophosphate synthetase. Anal Biochem 1978;89:355-359. 8 Braven J, Hardwell TR, Seddon R, Whittaker M. A spectrophotometric assay of phosphoribosyl pyrophosphate synthetase. Ann Clin Biochem 19&1;21:366-371. 9 Sakuma R, Nishina T, Kitamura M, Yamanaka H, Kamatani N, Nishioka K. Screening for adenine and hypoxanthine phosphoribosyltransferase deficiencies in human erythrocytes by high-performance liquid chromatography. Clin Chim Acta 1987;170:281-290. 10 Losman MJ, Hecher S, Woo S, Becker M. Diagnostic evaluation of phosphoribosyl pyrophosphate synthetase activities in hemolysates. J Lab Clin Med 1984;103:932-943. 11 Hardwell TR, Braven J, Shaw S, Whittaker M. Phosphoribosyl pyrophosphate synthetase and glutathione reductase in erythrocytes from hyperuricaemic and gout patients. Clin Chim Acta 1982;126:217-226. 12 Valentine WN, Fink K, Paglia DE, Harris SR, Adams WS. Hereditary hemolytic anemia with human erythrocyte pyrimidine 5’-nucleotidase deficiency. J Clin Invest 1974;54:866-879. 13 Van Kampen EJ, Zijlstra WG. Standardisation of haemoglobinometry II: the hemoglobin cyanide method. Clin Chim Acta 1961;6:538-544. 14 Fujimori S, Akaoka I, Sakamoto K, Yamanaka H, Nishioka K, Kamatani N. Common characteristics of mutant adenine phosphoribosyltransferase from four separate Japanese families with 2,8-dihydro~adenine urol~thiasis associated with partial enzyme deficiencies. Hum Genet 1985;71:171-176. 15 Sakuma R, Nishina T, Kitamura M. Deproteinizing methods evaluated for determination of uric acid in serum by reversed-phase liquid chromatography with ultraviolet detection. Clin Chem 1987;33:1427-1430. 16 Becker MA, Raivio KO, Bakay B, Adams WB, Nyhan WL. Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions. J Clin Invest 1980;65:109-120.
152 17 Paglia DE, Fink K, Valentine WN. Additional data from two kindreds with genetically induced deficiencies of erythrocyte pyrimidine nucleotidase. Acta Haematol 1980;63:262-267. 18 Fox IH, Kelley WN. Human phosphoribosylpyrophosphate synthetase. J Biol Chem 1972;247:21262131. 19 Becker MA, Meyer LJ, Huisman WH, Lazar C, Adams WB. Human erythrocyte phosphoribosylpyrophosphate synthetase. Subunit analysis and states of subunit association. J Biol Chem 1977;252:3911-3918. 20 Meyer LJ, Becker MA. Human erythrocyte phosphoribosylpyrophosphate synthetase. Dependence of activity on state of subunit association. J Biol Chem 1977;252:3919-3925. 21 Lachant NA, Zerez CR, Tanaka KR. Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5’nucleotidase deficiency. Biochim Biophys Acta 1989;994:81-88.
143
0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
CCA 05128
Phosphoribosylpyrophosphate synthetase in human erythrocytes: assay and kinetic studies using high-performance liquid chromatography Ryozo Sakuma ‘, Toshihiro Nishina ‘, Hisashi Yamanaka 2, Naoyuki Kamatani 2, Kusuki Nishioka 2, Masako Maeda 3 and Akio Tsuji 3 ’ Department of Clinical Chemistry, Toranomon Hospital, and Okinaka Memorial Institute for Medical Research, Minato-ku, Tokyo, 2 Institute of Rheumatology, Tokyo Women’s Medical College, Shinjuku-ku, Tokyo, and ’ School of Pharmaceutical Sciences, Showu University, Shinagawa-ku, Tokyo (Japan) (Received 30 January 1991; revision received 17 August 1991; accepted 19 August 1991) Key words: High-performance
liquid chromatography; Phosphoribosylpyrophosphate Pyrimidine 5’-nucleotidase deficiency
synthetase; Gout;
Summary A method using high-performance liquid chromatography (HPLC) for determination of phosphoribosylpyrophosphate (PRPP) synthetase activity in human erythrocytes has been developed and PRPP synthetase activity on purine and pyrimidine metabolic disorders has been studied. Kinetic properties of erythrocyte PRPP synthetase of patients with gout and of a patient with pyrimidine 5’nucleotidase deficiency were compared with those of healthy subjects. The mean of PRPP synthetase activity of gouty patients was a little higher (P < 0.01) than that of healthy subjects. The response of the enzyme for ATP of gouty patients was different from that of healthy subjects. The shapes of activation curve of the enzyme for inorganic phosphate were hyperbolic in gouty patients and in a patient with pyrimidine 5’-nucleotidase deficiency.
Introduction Phosphoribosylpyrophosphate synthetase (PRPP synthetase, EC 2.7.6.1) is a purine de novo enzyme. PRPP synthetase catalyzes the synthesis of PRPP from
Correspondence to: R. Sakuma, Department of Clinical Laboratory, Toranomon Branch Hospital, 1-3-1 Kajigaya, Takatsu-ku, Kawasaki 213, Japan.
144
ribose-j-phosphate (R5P) and adenosine5’-triphosphate (ATP) in the presence of inorganic phosphate and magnesium. The clinical importance of PRPP synthetase has been increased by the discovery of the inherited superactivity of the enzyme in red blood cells from patients with gout and overproduction of uric acid [1,2]. The methods for assay of PRPP synthetase in red blood cells have been studied by several workers in recent years [3-81. Many of the current methods for PRPP synthetase assay are radiochemical techniques [3-51. Such radiochemical techniques have been difficult to apply in clinical laboratories, due to the needs for special equipment for handling radioactive substances and for several complicated procedures. Spectrophotometric methods using enzyme-linked system are convenient, but these methods are hampered by poor sensitivity or poor precision [6-81. A method using high-performance liquid chromatography (HPLC) does not need special equipment or complicated procedures. We have already developed a nonradiochemical method using HPLC for assay of purine salvage enzymes, and have obtained good sensitivity and good precision [9]. We have therefore developed a method using HPLC for determination of PRPP synthetase activity in human erythrocytes, and have applied the method to study of PRPP synthetase activity on purine and pyrimidine metabolic disorders: gout, adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) deficiency, xanthine oxidase (X0, EC 1.2.3.2) deficiency, and pyrimidine 5’-nucleotidase (P5N, EC 3.1.3.5) deficiency. The mean of PRPP synthetase activity in erythrocytes from patients with gout was found to be the same level as that of healthy subjects [5,10], but an increase in the enzyme activity of patients with gout has been reported by Hardwell et al. [ll]. A decrease in the activity of PRPP synthetase in erythrocytes from patients with P5N deficiency has been reported by Valentine et al. [12]. However, the nature of the decreased PRPP synthetase activity in PSN-deficient erythrocytes remains unclear. Thus, we have studied kinetic properties of PRPP synthetase of gouty patients and a patient with P5N deficiency. Materials and methods Principle
PRPP synthetase catalyzes the following reaction: R5P + ATP + PRPP + AMP. Adenosine-5’-monophosphate (AMP) produced by the reaction is converted into adenosine-5’-diphosphate (ADP) with adenylate kinase (AK, EC 2.7.4.3): AMP + ATP -+ 2 ADP. The rate of the reaction catalyzed by PRPP synthetase is followed by measuring the increase in absorbance of ADP after separation of ADP from AMP and ATP by a reversed-phase HPLC.
145 Reagents
AMP, ADP, ATP (sodium salt), AK, R5P (sodium salt), and reduced glutathione were purchased from Boehringer Mannheim GmbH (Mannheim, FRG). Charcoal was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dextran (T-70) was from Pharmacia Fine Chemicals (Uppsala, Sweden). All other reagents were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sample preparation
Venous blood samples, obtained from 27 healthy subjects, 28 gouty patients, nine patients with APRT deficiency, a patient with X0 deficiency, and a patient with P5N deficiency, were collected in heparinised tubes. After centrifugation at 650 X g for 5 min at 4°C the plasma and buffy coat were removed. The remaining red cells were washed three times with 154 mmol/l NaCl, and packed by centrifugation at 650 X g for 10 min at 4°C. The packed cells were frozen and stored at -80’ C without loss of enzyme activity. The packed red cells were lysed with freeze-thawing ( - 80 and 37°C) twice and diluted with 5 volume of a charcoal-dextran suspension containing 10 g/l charcoal and 1 g/l dextran in cold distilled water. After mixing for about 10 s in a vortex mixer, the lysates were left for 10 min at 4°C and then centrifuged at 10,000 x g for 10 min at 4°C. The supernatant were used as enzyme samples. Hemoglobin (Hb) was determined by the cyanmethemoglobin method [13]. Enzyme assay
A reaction mixture contained 40 mmol/l sodium phosphate buffer, pH 7.4, 1.0 mmol/l R5P, 1.4 mmol/l ATP, 6 mmol/l MgCl,, 1 mmol/l reduced glutathione, and 1.8 IU/ml AK in a final volume of 2.0 ml. The reaction mixture was incubated for 5 min at 37°C. ATP was prepared fresh immediately before the enzyme assay. The enzyme reaction was started by the addition of 0.1 ml of the red cell lysate. After further incubation at 37 ’ C, 0.5 ml of the reaction mixtures were withdrawn at 10 min and 30 min into a tube containing 0.5 ml of 0.6 mol/l perchloric acid. After mixing for about 10 s, the mixtures were left for 5 min and centrifuged at 2,000 x g for 5 min. The supernatant, neutralized by adding the same volume of 0.4 mol/l Na,HPO,, were used as samples for HPLC analysis. Apparatus and chromatographic
conditions
The HPLC apparatus (from Tosoh, Co. Ltd., Tokyo, Japan) consisted of a Model 803D pump, an AS-48 autosampler (sample loop, 50 ~1) and a Model UV 8 II spectrophotometer connected to a Model CP-8000 chromatoprocessor. The spectrophotometer was set at 260 nm and 0.04 absorbance units full scale. AI1 separations were performed at ambient temperature on a column (25 cm x 4.6 mm i.d.1 packed with Tosoh ODS-120A gel (5 pm particle size) and a guard filter
146
(Tosoh line filter G). The mobile phase was sodium phosphate buffer (40 mmol/l, pH 5.6) containing 10 ml of methanol/l, It was continually degassed by a Erma Model ERC-3510 degasser (Erma Optical Works Ltd., Tokyo, Japan) connected to the 803D pump; the flow rate was 0.8 ml/min at 75 kg/cm*. ADP was determined by comparing the peak area in the chromatogram with values obtained from a standard curve. PRPP synthetase activity was calculated from the increase of ADP for 20 min. Results Chromatograms
of reaction mixtures
Figure 1 shows the chromatograms of the reaction mixtures after the enzyme reaction of erythrocyte PRPP synthetase from a healthy subject. The retention times after injection for ATP, ADP, and AMP were 7.5, 10, and 21 min, respectively. The peak of ADP was clearly separated from ATP and no peaks of unknown components, which would interfere with ADP peak, were observed with the progress of the enzyme reaction. Precision and sensitivity
The within-run precision of the method (n = 10) was evaluated by testing hemolysate of a healthy subject. Mean +_SD was 1.16 + 0.031 pmol/min per g Hb and the coefficient of variation (CV) was 2.7%. The product (ADP) concentration increased linearly with the reaction time for 40 min. ADP concentration in the reaction mixture after the enzyme reaction for 20 min was 60 pmol/l. The
(a>
(b)
ATP
DP
-f 0
--+ 10 Tir
20 ( inin
)
0
i
?I.-
10 Time
20 ( min
)
Fig. 1. HPLC profiles of the reaction mixtures after the enzyme reaction of erythrocyte PRPP synthetase from a healthy subject. (a) is the reaction mixture incubated for 10 min, and (b) is for 30 min. Elution conditions are described in the text.
147
sensitivity (detection limit) of the method evaluated by 2 SD of blank reaction was 0.005 pmol/min per g Hb. Comparison of a spectrophotometric method
This method was compared with the spectrophotometric method of Valentine et al. [6]. The within-run precision of the spectrophotometric method (n = 10) was evaluated by testing the same sample used in the HPLC method. Mean k SD was 1.03 + 0.053 pmol/min per g Hb and the CV was 5.1%. 2 SD of blank reaction was 0.07 pmol/min per g Hb. The activity of PRPP synthetase in erythrocytes from 38 outpatients was measured with both methods. Mean + SD obtained by the HPLC method was 1.04 + 0.09 pmol/min per g Hb and mean f SD obtained by the spectrophotometric method was 0.91 f 0.16 pmol/min per g Hb. PRPP sjrzthetase activities
The activity of PRPP synthetase obtained with erythrocytes from 27 healthy subjects (12 males and 15 females) was 1.10 + 0.11 pmol/min per g Hb (mean + SD). The present method was applied to assay of PRPP synthetase in erythrocytes from 28 patients with gout with normal uric acid excretion, nine patients with APRT deficiency, a patient with X0 deficiency, and a patient with P5N deficiency. The results are shown in Table I. The mean of PRPP synthetase activity of 28 patients with gout was a little higher (P < 0.01, t test) than that of 27 healthy subjects. The activities of PRPP synthetase of nine patients with APRT deficiency were a little higher than the mean of healthy subjects. The activity of the patient with xanthine oxidase deficiency was the lower limit of normal range. The activity of the patient with P5N deficiency was 66% of the mean of healthy subjects.
TABLE I PRPP synthetase activities in erythrocytes from patients with purine metabolic disorders and a patient with P5N deficiency Activity &mol/min
Diagnosis
per g Iib)
Mean + SD Normal (n = 27) Gout (n = 28) APRT deficiency a Homozygote (QO/QO) Heterozygote (QO/l) (n = 3) Japanese-type (J/J) (n = 5) X0 deficiency PSN deficiency ’ Letters in parentheses
1.10+0.11 1.28kO.11 1.30 1.13kO.02 1.16+0.12 0.96 0.73
indicate the genotypes of APRT deficiency [141.
148
, g
(a>
,
(b)
1.5
z .E c 1.0 2 2 .2‘ 0.5 .>
10
,
20
30
40
0
(b)
2
4
6
6
P G-mrol/l> % (mnol/l> Fig. 3. The relationships between the concentrations of activators, inorganic phosphate (a) and magnesium (b), and the activities of PRPP synthetase of a healthy subject (01, a gouty patient co), and the patient with P5N deficiency (A ).The enzyme reaction of (a) was carried out in 0.1 mol/l Tris-HC1 buffer, pH 7.4, containing 1.0 mmol/l RSP, 1.4 mmol/l ATP, 6 mmol/l MgCI,, 1 mmol/l reduced glutathione, 1.8 W/ml AK, and various concentrations of inorganic phosphate.
149
PSN-deficient patient were compared with those of the healthy subject (Fig. 3). The shapes of activation curves for inorganic phosphate were hyperbolic. Discussion The radiochemical methods for PRPP synthetase assay are based on the detection of isotopically labelled products separated from the substrates with high-voltage electrophoresis or thin-layer chromatography [3-51. The radiochemical techniques have required special equipment and several complicated procedures. Our method does not need special equipment and complicated procedures. AMP produced by the enzyme reaction is metabolized with other enzymes in erythrocytes, so AMP was converted into ADP with AK in our method. The treatment of the lysate with charcoal-dextran has been incorporated in the assay procedure to remove nucleotides in erythrocytes. Termination of the enzyme reaction and deproteinization of the solution after the enzyme reaction were simultaneously carried out with perchloric acid [El. Perchloric acid was immediately neutralized with sodium phosphate to avoid the hydrolysis of ATP in perchloric acid solution. A spectrophotometric method using the enzyme-linked system was reported by Valentine et al. [6], and modifications of the assay procedure of Valentine et al. were reported [7,81. These methods are based on measuring the decrease in absorbance of NADH, and require monitoring for 60 min at 340 nm. In addition, these methods require highly diluting sample to avoid the increase in initial absorbance of reaction solution. The sensitivity and precision of the methods are poor: 2 SD of brank reaction was 8% of normal PRPP synthetase activity [S] and the within-run CVs were 3-7% [7,8]. Our method is based on measuring the increase in absorbance of ADP at 260 nm. The molar extinction coefficient of ADP at 260 nm is greater than that of NADH at 340 nm, and the dilution of sample in our method is less than that in the spectrophotometric methods. In our method, 2 SD of brank reaction was 0.005 pmol/min per g Hb corresponding to 0.5% of normal value and the within-run CV was 2.7%. The data are good as compared with the results obtained by the spectrophotometric method of Valentine et al. [6]: 2 SD of brank reaction was 0.07 Fmol/min per g Hb and the within-run CV was 5.1%. The value of 1.04 f 0.09 pmol/min per g Hb obtained by our method is similar to the value of the spectrophotometric method (0.91 k 0.16 pmol/min per g Hb). The normal range of PRPP synthetase activity in erythrocytes obtained by our method was 1.10 k 0.11 pmol/min per g Hb. The value is similar to previously reported values: 0.97 + 0.27, 1.08 _t 0.30, and 1.50 f 0.25 pmol/min per g protein by radiochemical methods [4,5,101, 0.57 -t 0.07 and 1.60 f 0.52 pmol/min per g protein by spectrophotometric methods [7,8]. In our method, the mean of PRPP synthetase activity of gouty patients with normal excretion of uric acid was a little higher (P < 0.01) than that of healthy subjects (Table I). The result is different from the data previously reported [5,10]. Becker et al. [5] reported that the mean of PRPP synthetase activity of normal subjects was 1.08 + 0.30 pmol/min per g protein and the mean of gouty patients
1.50
with normal production of uric acid was 1.15 f 0.28 pmol/min per g protein. Losman et al. [lo] also reported that the activity of normal subjects (1.50 + 0.25 pmol/min per g protein) was the same level as gouty patients with normal uric acid excretion (1.45 + 0.23 pmol/min per g protein). However, Hardwell et al. [ll] have found out a significant increase 0’ < 0.05) of the activity of PRPP synthetase in gouty patients (2.03 f 0.74 pmol/min per g protein) compared with the values for control subjects (1.60 f 0.52 pmol/min per g protein). The differences of these results could be contributed to the concentration of ATP rather than R5P using as substrate, since the response of PRPP synthetase for ATP in the gouty patient was different from that of the healthy subject (Fig. 2b). PRPP synthetase of the gouty patient gave higher activity than that of the healthy subject at 1.33 mmol/l ATP used in our method and in the method of Hardwell et al. [ll], but gave the same activity level as the healthy subject at 0.5 mmol/l ATP used in the methods of Becker et al. [5] and of Losman et al. [lo]. The same results as shown in Fig. 2b were obtained in other gouty patients and other healthy subjects (data not shown). The apparent K, value for R5P of the gouty patient was similar to that of the healthy subject, but the increase of V,,, was observed (Fig. 3aI. The shape of activation curve of PRPP synthetase for inorganic phosphate was hyperbolic in the gouty patient (Fig. 3a). The hyperbolic activation for inorganic phosphate has been found in normal hemolysates chromatographed on Sephadex G-25 [161 or treated with activated charcoal [31, while the sigmoidal activation has appeared in crude [3] or dialyzed hemolysates [lo]. The shape of activation curve for magnesium of the gouty patient was similar to that of the healthy subject (Fig. 3b). A decrease in the activity of PRPP synthetase in erythrocytes of hereditary P5N deficiency has been reported by Valentine et al. [12]. Paglia et al. [17] reported the increase of the K, value for R5P of PRPP synthetase in PSN-deficient erythrocytes, about two- and three-fold of normal. In this study, the marked increase of K, value for R5P was not obtained in the patient with P5N deficiency. The differences of these results could be related to the treatment of the lysate with charcoal-dextran to remove nucleotides. Fox and Kelley [181 observed that 1 mmol/l cytidine di- and triphosphate inhibited PRPP synthetase activity at nonsaturating substrate concentrations. The accumulated pyrimidine nucleotides, which inhibit PRPP synthetase activity, were removed from the PSN-deficient erythrocyte by charcoal in our method, so the increase of the K, value for R5P of PRPP synthetase was not observed in the PSN-deficient erythrocyte. Although the accumulated pyrimidine nucleotides were removed by charcoal and the enzyme reaction was performed in the presence of saturating substrate concentrations, the decreased PRPP synthetase activity was obtained in the PSN-deficient erythrocyte. The decreased PRPP synthetase activity could not be caused by changes of the response of the enzyme for activators, inorganic phosphate and magnesium, because the shapes of activation curves for inorganic phosphate and magnesium of the PSN-deficient patient were similar to those of the healthy subject. The state of the subunit aggregation of PRPP synthetase is relation with the enzyme activity [19,201. The molecular size of PRPP synthetase of the PSN-deficient erythrocyte was compared with that of a healthy subject by gel filtration. The
151
result obtained by gel filtration was similar to the data reported by Lachant et al. [Zll (data not shown). The decreased PRPP synthetase activity of the PSN-deficient erythrocyte could be related to the amount of aggregated forms rather than the amount of disaggregated forms. However, further studies are needed to clarify the nature of the decreased PRPP synthetase activity in PSN-deficient erythrocytes. Acknowledgement The authors wish to thank Dr. Shiro Miwa for providing a blood sample of the patient with pyrimidine 5’nucleotidase deficiency. References 1 Becker MA, Meyer LJ, Wood AW, Seegmiller JE. Purine overproduction in man associated with increased phosphoribosylpyrophosphate synthetase activity. Science 1973;179:1123-1126, 2 Sperling 0, Eilam S, Persky-Brosh S, de Vries A. Accelerated erythrocyte 5-phosphoribosyl-l-pyrophosphate synthesis. A familial abnormality associated with excessive uric acid production and gout. Biochem Med 1972;6:310-316. 3 Hershko A, Razin A, Mager J. Regulation of the synthesis of 5-phosphoribosyl-I-pyrophosphate in intact red blood cells and in cell-free preparations. Biochim Biophys Acta 1969;184:64-76. 4 Fox IH, Kelley WN. Human phosphoribosylpyrophosphate synthetase. J Biol Chem 1971;246:57395748. 5 Becker MA, Meyer LJ, Wood AW, Seegmiller JE. Gout with purine overproduction due to increased phosphori~sylpyrophosphate synthetase activity. Am J Med 1973;55:232-242. 6 Valentine WN, Kurschner KK. Studies on human erythrocyte nucleotide metabolism. I Nonisotopic methodologies. Blood 197239666-673. 7 Ferrari M, Giacomello A, Salerno C, Messina E. A spectrophotometric assay for phosphoribosyl pyrophosphate synthetase. Anal Biochem 1978;89:355-359. 8 Braven J, Hardwell TR, Seddon R, Whittaker M. A spectrophotometric assay of phosphoribosyl pyrophosphate synthetase. Ann Clin Biochem 19&1;21:366-371. 9 Sakuma R, Nishina T, Kitamura M, Yamanaka H, Kamatani N, Nishioka K. Screening for adenine and hypoxanthine phosphoribosyltransferase deficiencies in human erythrocytes by high-performance liquid chromatography. Clin Chim Acta 1987;170:281-290. 10 Losman MJ, Hecher S, Woo S, Becker M. Diagnostic evaluation of phosphoribosyl pyrophosphate synthetase activities in hemolysates. J Lab Clin Med 1984;103:932-943. 11 Hardwell TR, Braven J, Shaw S, Whittaker M. Phosphoribosyl pyrophosphate synthetase and glutathione reductase in erythrocytes from hyperuricaemic and gout patients. Clin Chim Acta 1982;126:217-226. 12 Valentine WN, Fink K, Paglia DE, Harris SR, Adams WS. Hereditary hemolytic anemia with human erythrocyte pyrimidine 5’-nucleotidase deficiency. J Clin Invest 1974;54:866-879. 13 Van Kampen EJ, Zijlstra WG. Standardisation of haemoglobinometry II: the hemoglobin cyanide method. Clin Chim Acta 1961;6:538-544. 14 Fujimori S, Akaoka I, Sakamoto K, Yamanaka H, Nishioka K, Kamatani N. Common characteristics of mutant adenine phosphoribosyltransferase from four separate Japanese families with 2,8-dihydro~adenine urol~thiasis associated with partial enzyme deficiencies. Hum Genet 1985;71:171-176. 15 Sakuma R, Nishina T, Kitamura M. Deproteinizing methods evaluated for determination of uric acid in serum by reversed-phase liquid chromatography with ultraviolet detection. Clin Chem 1987;33:1427-1430. 16 Becker MA, Raivio KO, Bakay B, Adams WB, Nyhan WL. Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions. J Clin Invest 1980;65:109-120.
152 17 Paglia DE, Fink K, Valentine WN. Additional data from two kindreds with genetically induced deficiencies of erythrocyte pyrimidine nucleotidase. Acta Haematol 1980;63:262-267. 18 Fox IH, Kelley WN. Human phosphoribosylpyrophosphate synthetase. J Biol Chem 1972;247:21262131. 19 Becker MA, Meyer LJ, Huisman WH, Lazar C, Adams WB. Human erythrocyte phosphoribosylpyrophosphate synthetase. Subunit analysis and states of subunit association. J Biol Chem 1977;252:3911-3918. 20 Meyer LJ, Becker MA. Human erythrocyte phosphoribosylpyrophosphate synthetase. Dependence of activity on state of subunit association. J Biol Chem 1977;252:3919-3925. 21 Lachant NA, Zerez CR, Tanaka KR. Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5’nucleotidase deficiency. Biochim Biophys Acta 1989;994:81-88.
