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Inosinic Acid Dehydrogenase Activity in the Lesch-Nyhan Syndrome

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Inosinic Acid Dehydrogenase Activity in the Lesch-Nyhan Syndrome Inosinic acid dehydrogenase activity in the Lesch-Nyhan syndrome D. Michael Pehlke, … , Edward W. Holmes, William N. Kelley J Clin Invest. 1972;51(6):1398-1404. https://doi.org/10.1172/JCI106935. Research Article Inosinic acid dehydrogenase was evaluated in normal subjects and in patients with the Lesch-Nyhan syndrome. A significant difference in activity was found between erythrocytes derived from normal controls (1.21±0.47 pmoles/hr per mg protein) and from 15 patients with the Lesch-Nyhan syndrome (6.72±6.23 pmoles/hr per mg protein). However, no difference in activity was demonstrable in muscle or leukocytes derived from normal and Lesch-Nyhan patients. The increased activity of inosinic acid dehydrogenase in erythrocytes from patients with the Lesch-Nyhan syndrome is due to stabilization of the enzyme in vivo as well as the absence of an inhibitor which is present in erythrocytes from normal subjects. Find the latest version: https://jci.me/106935/pdf Inosinic Acid Dehydrogenase Activity in the Lesch-Nyhan Syndrome D. MICHAEL PEHLKE, JOHN A. MCDONALD, EDWARD W. HOLMES, and WILLIAM N. KELLEY From the Division of Rheumatic and Genetic Diseases, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 A B S T R A C T Inosinic acid dehydrogenase was evalu- normalities have been consistently described in patients ated in normal subjects and in patients with the Lesch- with the Lesch-Nyhan syndrome. These include an Nyhan syndrome. A significant difference in activity accelerated rate of purine biosynthesis (2), increased in- was found between erythrocytes derived from normal tracellular levels of PP-ribose-P in both erythrocytes controls (1.21±0.47 pmoles/hr per mg protein) and (3) and cultured skin fibroblasts (4), and increased from 15 patients with the Lesch-Nyhan syndrome (6.72 activity of the enzyme adenine phosphoribosyltransfer- ±6.23 pmoles/hr per mg protein). However, no differ- ase in erythrocytes (1, 5). All of these effects may be ence in activity was demonstrable in muscle or leuko- interpreted as secondary manifestations of the de- cytes derived from normal and Lesch-Nyhan patients. ficiency of hypoxanthine-guanine phosphoribosyltrans- The increased activity of inosinic acid dehydrogenase ferase (5). in erythrocytes from patients with the Lesch-Nyhan In this paper we describe a significant and con- syndrome is due to stabilization of the enzyme in vivo sistent elevation of activity of the enzyme inosinic acid as well as the absence of an inhibitor which is present (lehydrogenase in erythrocyte lysates from patients with in erythrocytes from normal subjects. the Lesch-Nyhan syndrome. Inosinic acid dehydrogenase (IMP: NAD' oxidoreductase, E.C. 1.2.1.14) catalyzes the nicotine adenine dinucleotide (NAD+) -dependent INTRODUCTION conversion of IMP to xanthosine-5'-monophosphate Individuals with the Lesch-Nyhan syndrome exhibit a (XMP) and represents the first reaction unique to the functional deficiency of hypoxanthine-guanine phos- synthesis de novo of GMP from IMP. This enzyme, phoribosyltransferase (HGPRT)' which is necessary which had not previously been assayed in human tis- for the phosphoribosylpyrophosphate (PP-ribose-P)-de- sues, should play a critical role in the Lesch-Nyhan pendent formation of inosine-5'-monophosphate (IMP) syndrome. In the absence of hypoxanthine-guanine and guanosine-5'-monophosphate (GMP) from the pu- phosphoribosyltransferase activity, the pathway initiated rine bases hypoxanthine and guanine, respectively (1). by IMP dehydrogenase represents the only known Clinically, the defect is manifested by hyperuricemia mechanism for the production of guanine nucleotides. and hyperuricaciduria, self-mutilation, choreoathetosis, Several different factors appear to be responsible for spasticity, and mental retardation (2). the observed increase in enzyme activity. In addition to a deficiency of hypoxanthine-guanine phosphoribosyltransferase, several other biochemical ab- METHODS Hypoxanthine-8-'4C (4.2, 52.0, and 60.1 mCi/mmole) was Received for publication 21 October 1971 and in revised obtained from New England Nuclear Corp., Boston, Mass., form 5 January 1972. Schwarz Bio Research Inc., Orangeburg, N. Y., and Amer- 1 Abbreviations used in this paper: GMP, guanosine-5'- sham/Searle Corp., Arlington Heights, Ill., respectively. monophosphate; HGPRT, hypoxanthine-guanine phospho- Nicotine adenine dinucleotide (NAD1) was purchased from ribosyltransferase; IMP, inosine-5'-monophosphate; NAD+, Sigma Chemical Company, St. Louis, Mo. The dimagnesium nicotine adenine dinucleotide; PP-ribose-P, phosphoribosyl- salt of PP-ribose-P and the tetra sodium salt of PP-ribose-P pyrophosphate; XMP, xanthosine-5'-monophosphate. were obtained from P-L Biochemicals, Inc., Milwaukee, 1398 The Journal of Clinical Investigation Volume 51 1972 Wis., and Sigma, respectively. All other reagents were of thawing twice. The hemolysates were then dialyzed in 0.05 the highest purity commercially available. M sodium phosphate, pH 7.4, for 2 hr and assayed. Five patients (E. S., J. K., W. E., D. C., and D. G.) with Inosinic acid dehydrogenase activity was determined by the Lesch-Nyhan syndrome were admitted to the Clinical Re- quantitating the production of xanthosine monophosphate search Center at Duke Hospital where they were maintained (XMP)-'4C utilizing modifications of the assays developed on a diet essentially free of purines containing 1800 k-cal by Salser and Balis (8) and Saccocia and Miech (9). The and 70 g protein. The only medications were allopurinol and assay was optimized with regard to substrate concentration diazepam which were found to have no effect on inosinic and cation requirement and contained 0.01 Amoles IMP-14C, acid dehydrogenase levels. Hemolysates were also obtained 0.1 Amoles NAD', 10 gmoles KCl, 4 Amoles sodium phos- from 10 other patients with the Lesch-Nyhan syndrome. phate buffer, pH 7.4, and the specified amount of the enzyme These samples were stored at - 20'C for varying periods preparation to be assayed in a total volume of 100 pl. The of time before assay. reaction was initiated by placing the tubes containing the Blood obtained by venipuncture was twice washed with reaction mixture in a water bath at 370C. After incubation normal saline and centrifuged at 1200 g for 10 min. The for 2 hr at 370C the reaction was stopped by the addition washed, packed erythrocytes were frozen at - 200C, thawed, of 100 1l of cold 95% ethanol. Protein was removed by and the resulting hemolysate dialysed against 0.05 M sodium centrifugation at 1200 g for 15 min. 50 pl of the supernate phosphate buffer, pH 7.4, for 2 hr at 40C. All hemolysates was spotted on Whatman DE-81 paper with 0.1 umoles of obtained from normal volunteers were prepared in a similar carrier XMP and developed in an ascending system for 4 manner and assayed within 2 wk unless otherwise specified. hr in 0.2 M ammonium formate buffer, pH 5.0 (R,: XMP, Muscle tissue from patients with the Lesch-Nyhan syn- 0.32; IMP, 0.72). After development, the spot containing drome was obtained by biopsy while muscle tissue from XMP was detected with an ultraviolet light source, cut subjects with normal HGPRT activity was obtained either out, and counted at 63% efficiency in a Packard Tri-Carb by biopsy or at autopsy. Enzyme activity was comparable liquid scintillation counter (Packard Instrument Co., Inc., in muscle obtained by either technique. Muscle tissue was Downers Grove, Ill.). Guanosine monophosphate (GMP) homogenized in 10 vol of 0.05 M sodium phosphate buffer, was separated from IMP and XMP by high voltage electro- pH 7.4. The homogenate was centrifuged at 13,000 g for phoresis in 0.05 M sodium citrate buffer, pH 3.53. Identity 30 min and the supernate dialyzed in 0.05 M sodium phos- of XMP was confirmed by ascending chromatography in phate buffer for 4 hr at 4°C. ethanol: 1.0 M ammonium acetate, pH 5.0 70: 30; (Rr: 0.11) Leukocytes were prepared by a modification of the method and high voltage electrophoresis on Whatman 3MM paper of Chodirker, Bock, and Vaughn (6). Venous blood (25 ml) in both 0.05 M sodium borate, pH 8.8, and 0.05 M sodium was drawn and immediately mixed with 475 ml of 0.85% citrate, pH 3.53. XMP production, under these assay condi- NaCl with 0.1% bovine serum albumin. After centrifuga- tions, was linear with respect to incubation time from 15 tion at 800 g for 30 min, the supernate was discarded and min to 3 hr and protein concentration from 1.25 to 12.5 the pellet mixed with 100 ml of 0.2% NaCl for 20 sec and mg/0.10 ml using crude hemolysate. then restored to isotonicity by the addition of 100 ml of Adenine phosphoribosyltransferase and HGPRT activity 1.61%o NaCl. The cells were centrifuged at 400 g for 10 min were assayed by the method of Kelley, Rosenbloom, Hender- and the lysis procedure was repeated once. The cells ob- son, and Seegmiller (10). tained from this procedure were washed twice in cold iso- IMP-8-14C of sufficient purity and specific radioactivity tonic saline. After the final wash the white cell pellet was for this study was not commercially available. Therefore, dissolved in 0.01 M Tris buffer pH 7.4 (80 mg wet weight IMP-8-14C was enzymatically synthesized from hypoxanthine- per ml). The cells were lysed by freezing and thawing 8-14C using a partially purified preparation of HGPRT twice in a dry ice and acetone bath. All leukocyte prepara- prepared as described below. The reaction mixture contained tions were assayed for inosinic acid dehydrogenase within 1.0 Amoles of hypoxanthine-8-'4C, 2.0 Amoles of PP-ribose-P, 24 hr of venipuncture. 5 Amoles of Mg", 10 gmoles of Cl-, 0.08 Amoles of Tris Erythrocytes were fractionated according to their density HCl, pH 7.4, and 1.6 mg of partially purified human after the method of Danon and Marikovsky (7).
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