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Hypoxanthine-Guanine Phosphoribosyltransferase: Characteristics of the Mutant Enzyme in Erythrocytes from Patients with the Lesch-Nyhan Syndrome

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Hypoxanthine-Guanine Phosphoribosyltransferase: Characteristics of the Mutant Enzyme in Erythrocytes from Patients with the Lesch-Nyhan Syndrome Hypoxanthine-guanine phosphoribosyltransferase: characteristics of the mutant enzyme in erythrocytes from patients with the Lesch-Nyhan syndrome William J. Arnold, … , Jean C. Meade, William N. Kelley J Clin Invest. 1972;51(7):1805-1812. https://doi.org/10.1172/JCI106982. Research Article The Lesch-Nyhan syndrome is characterized clinically by choreoathetosis, spasticity, selfmutilation, and mental and growth retardation. Biochemically, there is a striking reduction of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in affected individuals. We have examined erythrocytes from 14 patients with the Lesch-Nyhan syndrome for the presence of hypoxanthine-guanine phosphoribosyltransferase activity and enzyme protein. In contrast to the usual finding of no detectable hypoxanthine-guanine phosphoribosyltransferase activity, we have found low levels (0.002-0.79 nmoles/mg protein per hr) of hypoxanthine-guanine phosphoribosyltransferase activity in erythrocyte lysates from five of these patients. In three of the five patients, hypoxanthine-guanine phosphoribosyltransferase activity appeared to be substantially more labile in vivo than normal using erythrocytes which had been separated according to their density (age). Immunochemical studies using a monospecific antiserum prepared from a homogeneous preparation of normal human erythrocyte hypoxanthine-guanine phosphoribosyltransferase revealed immunoreactive protein (CRM) in hemolysate from all 14 patients with the Lesch-Nyhan syndrome. The immunoreactive protein from each patient gave a reaction of complete identity with normal erythrocyte hypoxanthine-guanine phosphoribosyltransferase and was present in quantities equal to those observed in normal erythrocytes. In addition, a constant amount of CRM was found in erythrocytes of increasing density (age) from patients with the Lesch-Nyhan syndrome despite the decreasing hypoxanthine-guanine phosphoribosyltransferase activity. These studies confirm previous data which indicate that the mutations leading to the Lesch-Nyhan syndrome are usually, if not […] Find the latest version: https://jci.me/106982/pdf Hypoxanthine-Guanine Phosphoribosyltransferase: Characteristics of the Mutant Enzyme in Erythrocytes from Patients with the Lesch-Nyhan Syndrome WILLIAM J. ARNOLD, JEAN C. MEADE, 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 ABSTRA CT The Lesch-Nyhan syndrome is charac- are usually, if not always on the structural gene coding terized clinically by choreoathetosis, spasticity, self- for hypoxanthine-guanine phosphoribosyltransferase. In mutilation, and mental and growth retardation. Bio- addition, although the mutant proteins appear to be chemically, there is a striking reduction of hypoxanthine- present in normal amounts, they are often very labile guanine phosphoribosyltransferase (HGPRT) activity in vivo with respect to enzymatic activity. These obser- in affected individuals. We have examined erythrocytes vations suggest that therapy directed at stabilization or from 14 patients with the Lesch-Nyhan syndrome for activation of enzyme activity in vivo may be of potential the presence of hypoxanthine-guanine phosphoribosyl- benefit. transferase activity and enzyme protein. In contrast to the usual finding of no detectable hypoxanthine-guanine phosphoribosyltransferase activity, we have found low INTRODUCTION levels (0.002-0.79 nmoles/mg protein per hr) of hypox- The Lesch-Nyhan syndrome is a bizarre, X-linked dis- anthine-guanine phosphoribosyltransferase activity in ease characterized by spasticity, choreoathetosis, self- erythrocyte lysates from five of these patients. In three mutilation, mental and growth retardation as well as of the five patients, hypoxanthine-guanine phosphori- hyperuricemia and hyperuricaciduria (1) which is due bosyltransferase activity appeared to be substantially to a striking reduction of hypoxanthine-guanine phos- more labile in vivo than normal using erythrocytes which phoribosyltransferase (HGPRT)' activity (2). The dis- had been separated according to their density (age). covery of a patient with the classical clinical syndrome in Immunochemical studies using a monospecific anti- whom the reduced hypoxanthine-guanine phosphoribosyl- serum prepared from a homogeneous preparation of nor- transferase activity was due to an altered affinity for mal human erythrocyte hypoxanthine-guanine phosphori- both substrates provided the first evidence for genetic bosyltransferase revealed immunoreactive protein (CRM) heterogeneity in the Lesch-Nyhan syndrome and demon- in hemolysate from all 14 patients with the Lesch-Nyhan strated that the mutation at least in this individual was syndrome. The immunoreactive protein from each pa- on the structural gene coding for this enzyme (3). Fur- tient gave a reaction of complete identity with normal ther studies of the mutant hypoxanthine-guanine phos- erythrocyte hypoxanthine-guanine phosphoribosyltrans- phoribosyltransferase in cultured skin fibroblasts derived ferase and was present in quantities equal to those ob- from affected patients provided further evidence for served in normal erythrocytes. In addition, a constant the existence of substantial genetic heterogeneity among amount of CRM was found in erythrocytes of increasing patients with the syndrome (4). density (age) from patients with the Lesch-Nyhan syn- In the present study, we have demonstrated the pres- drome despite the decreasing hypoxanthine-guanine ence of a low level of hypoxanthine-guanine phosphori- phosphoribosyltransferase activity. bosyltransferase activity in circulating erythrocytes from These studies confirm previous data which indicate that the mutations leading to the Lesch-Nyhan syndrome 'Abbreviations used in this paper: CRM, cross-reactive material; GMP, guanosine monophosphate; HGPRT, hy- Received for publication 22 November 1971 and in re- poxanthine-guanine phosphoribosyltransferase; PP-ribose-P, vised form 4 February 1972. tetrasodium phosphoribosylpyrophosphate. The Journal of Clinical Investigation Volume 51 July 1972 1805 some patients with the Lesch-Nyhan syndrome. The hemolysate is estimated to contain 140 Ag/ml of catalytically studies to be presented not only confirm the heterogeneity active hypoxanthine-guanine phosphoribosyltransferase pro- of the mutations on the structural gene coding for tein based on the recovery of hypoxanthine-guanine phospho- hy- ribosyltransferase activity and protein from this purification. poxanthine-guanine phosphoribosyltransferase in patients Antiserum was produced in fully grown white male rab- with the Lesch-Nyhan syndrome but also describe an bits against each of the highly purified hypoxanthine-guanine additional manifestation of the mutation in some pa- phosphoribosyltransferase isoenzymes as well as a partially tients: an accelerated half-life of enzyme activity in cir- purified (491 X) preparation of hypoxanthine-guanine phos- phoribosyltransferase. Initially separate rabbits received 0.23, culating erythrocytes. Despite the rapid loss of enzyme 0.45, and 0.53 mg of electrophoretic variant I, II, and III, activity the mutant protein itself appears to exhibit nor- respectively, which had been emulsified in Freund's complete mal stability. adjuvant and injected intradermally in the foot pads and neck. 1 month later the rabbits received a similar injection. Those receiving the partially purified preparation initially METHODS were injected in similar sites with 0.9 mg of protein emulsi- Hypoxanthine-8-'4C (3.07 mCi/mmole) and guanine-8-'4C fied in Freund's complete adjuvant. Blood was obtained by (6.05 mCi/mmole and 60 mCi/mmole) were purchased from laceration of the marginal ear vein and was allowed to sit New England Nuclear Corp., Boston, Mass. Tetrasodium at 4'C for 24 hr. After centrifugation at 10,000 g for 15 phosphoribosylpyrophosphate (PP-ribose-P) was purchased min at 4VC the antiserum was removed, divided into 1 ml from Sigma Chemical Co., St. Louis, Mo. Purified Noble portions, and stored at - 70'C. Agar and Freund's complete adjuvant were purchased from Direct inhibition of hypoxanthine-guanine phosphoribosyl- Difco Laboratories, Detroit, Mich. Coomassie blue was pur- transferase by antiserum was demonstrated by the addition chased from Mann Research Labs, Inc., New York. Methyl of a constant amount of heterospecific antiserum (50-250 1ul) phthalate and di-n-butyl phthalate came from Eastman to an equal volume of serial dilutions of dialyzed hemolysate Chemical Products, Inc., Kingsport, Tenn. All other re- followed by incubation at 37'C for 30 min and then at 4VC agents were of the highest quality commercially available. for 16 hr. After centrifugation at 5000 g for 20 min at 4VC, Hypoxanthine-guanine phosphoribosyltransferase was as- the supernatant was assayed for hypoxanthine-guanine phos- sayed by a previously described radiochemical technique (5). phoribosyltransferase activity. Incubation of hemolysate with All hemolysates were dialyzed at 4VC against 1 mmi Tris serum obtained from the same rabbits before immunization buffer pH 7.4 for 2 hr before being assayed. Under the and processed in a fashion identical to the antiserum did not usual assay conditions the final reaction mixture contained inhibit hypoxanthine-guanine phosphoribosyltransferase ac- 50 mmi Tris, 5 mm MgCl2, 1 mm PP-ribose-P, 0.1 mM purine tivity. A low level of hypoxanthine-guanine phosphoribosyl- base, and enzyme
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