89. 355-359 (1978)
A Spectrophotometric Assay for Phosphoribosylpyrophosphate Synt hetase M. FERRARI, A. GIACOMELLO,
AND E. MESSINA
Institute of Biological Chemistry and Institute of Rheumatology, University of Rome, and Center of Molecular Biology of C. N. R., Rome, Italy Received February 3, 1978; accepted April 7, 1978 A simple, rapid, and sensitive spectrophotometric assay of phosphoribosylpyrophosphate synthetase activity is described. This assay is based on the quantitative measurement of the reaction product AMP by a NADH-coupled enzyme method.
The enzyme Sphosphoribosyl1-pyrophosphate synthetase (PRPP synthetase),’ (EC 126.96.36.199) catalyzes the synthesis of PRPP and adenosine monophosphate (AMP) from ribose Sphosphate and adenosine 5’-triphosphate (ATP) in the presence of magnesium ions and inorganic phosphate. The description of increased PRPP synthetase activity in the erythrocytes from some gouty subjects has emphasized the need for a simple and sensitive assay for this enzyme (1). Most of the currently used assays for PRPP synthetase involve 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. The corresponding isotopically labeled nucleotide (end product of the reaction) is separated by means of techniques such as high-voltage electrophoresis (2) or paper or thin-layer chromatography
These methods are rather complex and expensive and require scintillation equipment which is not usually available in a clinical laboratory. The present paper describes a rapid, sensitive, and simple spectrophotometric assay of PRPP synthetase activity. * Abbreviations used: PRPP, phosphoribosylpyrophosphate; ATP, adenosine 5’-triphosphate; ADP, adenosine 5’-diphosphate; AMP, adenosine 5’-monophosphate; PEP, phosphoenol pyruvate; EDTA, ethylendiamine tetraacetate: MK, myokinase; PK. pyruvate kinase; LDH, lactate dehydrogenase.
0003-2697/78/0892-0355$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
All enzymes were commercial suspensions in 3.2 M (NH&SO4 from Boehringer A. G. Other reagents were high-purity commercial samples from Boehringer A. G., Merck A. G., and Sigma, Inc. Methods
PRPP synthetase was assayed in dialyzed erythrocyte lysates prepared from human heparinized venous blood according to published procedures (4). Erythrocytes, separated from the plasma by centrifugation, were washed twice in isotonic saline and dialyzed for 120 min against 8.0 mM sodium phosphate buffer, pH 7.4, containing 1 mM ethylendiamine tetraacetate (EDTA) and 5 mM mercaptoethanol. Protein in hemolysates were measured by the method of Lowry et al. (5). PRPP synthetase was assayed by a two-step procedure: First, the PRPP synthetase-catalyzed reaction was conducted at 37°C and was terminated by addition of perchloric acid; second, the reaction product, AMP, was then measured spectrophotometrically as previously described (6). Step I: PRPP synthetase-catalyzed reaction. The reaction mixture designed after published procedures (4,7) contained 0.5 mM ATP, 0.35 mM ribose j-phosphate, 2.5 mM mercaptoethanol, 1 mM EDTA, 5 mM MgCl*, 32 mM sodium phosphate, 50 mM Tris-HCl, pH 7.4, and from 0.37 to 1.87 mg/ml of protein from dialyzed hemolysate. All incubations were conducted at 37”C, and hemolysate was added last to initiate the reaction. A 2-ml volume of the reaction mixture was added at different periods of time to 2 ml of 1 M ice-cold perchloric acid. The precipitated protein was removed by centrifugation. A 2.5-ml volume of the supernatant was neutralized by adding 0.5 ml of a solution containing 0.5 M triethanolamineHCl and 2.0 M K&OS. After incubation in an ice bath for 10 mitt, the potassium perchlorate precipitate was removed by centrifugation. To avoid the MgNH,PO, precipitation during the spectrophotometric measurement carried out in Step 2, owing to the presence of (NH&SO4 in the most commonly available enzyme preparations of MK, PK, and LDH, the inorganic phosphate present in 2 ml of the supernatant was precipitated by adding 40 ~1 of 4 M NH&l and 50 ~1 of 2.5 M MgC&. After incubation at 4°C for 1 hr the precipitated salt was removed by centrifugation, and the supernatant was used for the quantitative determination of AMP. Step 2: AMP assay. AMP was measured by the following NADH-linked method: MK AMP + ATP-
PK, Mg2+, K+ 2 ADP + 2 PEP > 2 ATP + 2 pyruvate LDH 2 pyruvate
+ 2 NADH
+ 2 H+-
2 lactate + 2 NAD+
The reaction mixture contained 1 ml of the supernatant obtained in Step 1, lpmol of PEP, 0.25 pmol of NADH, 130 pmol of KCI, 3 IU of LDH, and 3 IU of PK in a final volume of 1.17 ml. ATP and Mg2+ ions necessary for the reaction were present in the supernatant obtained from Step 1 (1.1 X lO-4 M < [ATP] < 1.7 X lO-4 M; [Mg2+] = 4.9 X lO-2 M). The first spectrophotometric reading was made at 340 nm to determine the initial absorbance. A lo-p1 volume of 500 IU/ml of MK was then added to the mixture, and the reaction was followed to its completion at 340 nm. The reaction went to completion after 30 min at 25°C. AMP concentration was calculated from the optical density variation at 340 nm, assuming a molar extinction coefficient of 6220 for NADH. RESULTS
As previously described (6) 2 mol of NAD are formed from 1 mol of AMP when the procedure described in Step 2 is applied to AMP solutions of known concentration ranging from 0.5 to 5 x 10-j M. No difference in the relationship between [AMP] and optical density variation at 340 nm was observed when AMP (ranging from 1.2 to 12 x lo-” M) was added to the reaction mixture for PRPP synthetase assay in the absence of hemolysate and all procedures described in Steps 1 and 2 were carried out. Furthermore AMP does not break down significantly during incubation with the amounts of hemolysate employed for PRPP synthetase assay, and PRPP
1. Progress curve of the PRPP synthetase reaction. in the PRPP synthetase assay was 1.5 m&ml.
FERRARI ET AL.
FIG. 2. PRPP synthetase activity as a function of hemolysate concentration. Optical density variation at 340 nm in 60 min as a function of hemolysate protein concentration.
(product of PRPP synthetase reaction) at concentrations lower than 5 x lop4 M does not significantly influence the AMP assay (6). Under the assay conditions described, the PRPP synthetase reaction followed a linear course during the first 70 min (Fig. 1); the slope of the straight line describing the reaction progress was a linear function of the hemolysate protein concentration (Fig. 2). Control experiments without ribose 5-phosphate or ATP displayed no activity. The presence of PRPP synthetase in the enzyme system used for the AMP assay can be excluded since no activity was observed with acid-denaturated hemolysate. The accuracy of the PRPP synthetase activity determination on the same blood sample was ?7%. The mean hemolysate PRPP synthetase activity in 15 healthy adult males (mean age, 29 years) was 34 + 4 (SD) nmol/hr/mg of protein. This result is comparable to those obtained by other authors using isotopic procedures (4,8,9). The present assay for PRPP synthetase activity is rapid and simple and eliminates the expense and equipment requirements of radioisotopic assays. This method and the spectrophotometric assay for the hypoxanthineguanine phosphoribosyltransferase activity previously reported (10) can be used in the clinical laboratory, in which scintillation equipment is not usually available, to diagnose the specific genetic enzyme defects leading to primary hyperuricemia and gout described so far (1). REFERENCES 1. Wyngaarden, J. B., and Kelley, W. N. (1976) in Gout and Hyperuricemia, Grune & Stratton, New York. 2. Fox, I. H., and Kelley, W. N. (1971) J. Bid. Chem. 246, 5739-5748.
ASSAY FOR PRPP SYNTHETASE
3. Hershko, A., Razin, A., and Mager, 1. (1969) Biochim. Biophys. Acfa 184, 64-76. 4. Becker, M. A., Mayer, L. J., and Seegmiller, J. E. (1973) Amer. J. Med. 55, 232-242. 5. Lowry, 0. H., Rosebrough, N. J.. Farr, A. L., and Randall, R. J. (1951)5. Biol. Chem. 193, 265-275.
Salerno. C., and Giacomello, A. (1977) Anal. Biochem. 82, 204-209. 7. Sperling, O., Boer, P., Persky-Brosh, S., Kanazek, E., and De Vries, A. (1972) Rev.
8. Sperling, 0.. Persky-Brosh, S., Boer, P., and De Vries, A. (1974) in Purine Metabolism in Man (Sperling, 0.. De Vries, A., and Wyngaarden, J. B., eds.), pp. 299-305, Plenum Press, New York. 9. Tax. W. J. M.. and Veerkamp, J. H. (1977) C/in. Chim. Arra 78, 209-216. 0. Giacomello, A., and Salerno, C. (1977) Anal. Biochem. 79, 263-267.