D. T. Keough

Xanthine-containing calculi during allopurinol therapy.

We report a case of urate overproduction owing to the Lesch-Nyhan syndrome (deficiency of hypoxanthine-guanine phosphoribosyltransferase). Urate crystalluria was controlled by allopurinol therapy but renal calculi developed, which contained a variety of purines, particularly the relatively insoluble xanthine, as well as oxypurinol and hypoxanthine. The potential hazard from the increased amounts of xanthine that are produced during allopurinol therapy for urate overproduction is stressed, as well as the importance of maintaining a high urine flow rate even during such therapy.

Human hypoxanthine—guanine phosphoribosyltransferase. Development of a spectrophotometric assay and its use in detection and characterization of mutant forms

A simple and rapid spectrophotometric assay for the estimation of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in human tissues is described. It is based on the increase in absorbance at 257.5 nm which occurs when the substrate guanine is converted to its 5-mononucleotide, GMP. The assay has been developed to measure HGPRT activity in erythrocyte and lymphocyte lysates and in brain homogenates, and has been used in the screening of patients with hyperuricaemia and/or hyperuricosuria for HGPRT deficiency. It has also been used to determine the steady-state kinetic constants of a mutant form of the enzyme. The spectrophotometric assay is compared with the radioactive assay currently used to measure HGPRT activity.

Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme

A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7. Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with [35S]methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis. Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates. Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources. The Km values of recombinant HPRT for the substrates 5-phospho-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT.

Biochemical basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in nine families

SummaryThe concentration of hypoxanthine-guanine phosphoribosyltransferase (HPRT) cross-reacting material (CRM) was determined in haemolysates and/or lymphoblast lysates from nine patients with complete or partial deficiency of HPRT activity. Two of the patients had the fully developed Lesch-Nyhan syndrome and although they had undetectable HPRT activity, small amounts of CRM were found. HPRT-specific mRNA was not detected in lymphoblast lysates from one of these patients, while lysates from the other had a much reduced concentration. Samples from three patients with <0.1% of normal HPRT activity but with minor or no neurological manifestations were also found to contain small amounts of CRM. The other four patients whose HPRT activities ranged from 3 to 10% of normal were found to have CRM concentrations which varied from 26 to 100% of normal. In one patient with a partial deficiency theKm for 5-phospho-α-d-ribosyl-1-pyrophosphate was five times normal.

Localization of the 5-phospho-α-d-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase

Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.

HPRT-Deficiency associated with normal PRPP concentration and APRT activity

Deficiencies of HPRT are usually associated with increased concentrations of PRPP and increased levels of APRT activity in erythrocytes. We report the case of a male with a partial deficiency of HPRT in whom these two parameters were normal. The clinical features of this patient were those associated with severe hyperuricaemia and gout. Studies of intact erythrocytes showed rates of incorporation of [14C]hypoxanthine and of [14C]adenine into purine nucleotides which were almost indistinguishable from normal. However, HPRT activity in erythrocyte lysates was only 9% of normal. In cell extracts of cultured lymphoblasts, the HPRT activity was 20% of control values and the APRT activity was normal. The PRPP concentration and the rate ofde novo purine synthesis in cultured lymphoblasts were both intermediate between controls and lymphoblasts from patients with the Lesch-Nyhan syndrome.

Role of cysteine and lysine residues in human hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a purine salvage enzyme which is widely distributed in human tissues. It catalyses the synthesis of the purine mononucleotides, IMP and GMP. In humans, partial enzyme deficiency is associated with gout and hyperuricemia while complete enzyme deficiency results in the Lesch-Nyhan syndrome.

73 IS HPRT-LIKE PROTEIN PRESENT IN LESCH-NYHAN PATIENTS?

A complete deficiency of hypoxanthine-guanine phosphoribosyl-transferase (HPRT) activity results in the Lesch-Nyhan syndrome. Of the 15 Lesch-Nyhan patients studied by Wilson et al. (1), HPRT cross-reactinq material (CRM) was detected in lymphoblasts from only four (1.3%, 50%, 72% and 92% of control concentrations) whereas 12 had HPRT-specific mRNA. None of the three patients lacking mRNA had detectable CRM. We have studied CRM levels in haemolysates and lymphoblast lysates from two Lesch-Nyhan patients (R.W. and J.G). In the first series of experiments, normal CRM levels were measured and lysates from both patients gave strong precipitation lines in immunodiffusion analysis. In the second series, using a different HPRT preparation in the radioimmunoassay (RIA), very low CRM levels (0.5% and 0.1%) were obtained, although strong precipitation lines were still observed on Ouchterlony plates.A possible explanation for these discrepancies lies in a change in structure of normal HPRT which we have observed on storage, leading to a reduction in binding affinity for the antibody. The enzyme used in the first series of CRM determinations had been stored for an extended period, whereas enzyme used in the second series was freshly prepared. This explanation implies that the protein present in Lesch-Nyhan patients binds to the antibody with a lower affinity than does the normal enzyme (see also ref.2). These studies suggest that HPRT-like protein may be present in a higher percentage of Lesch-Nyhan patients than previously thought and may be present even when HPRT-specific mRNA cannot be detected, as in patient R.W.1. Wilson, J.M., Stout, J.T., Palella, T.D., Davidson, B.L., Kelley, W.N. and Caskey, C.T. (1986) J.Clin.Invest. 77:188–195.2. Wilson, J.M., Baugher, B.W., Landa. L. and Kelley, W.N. (1981) J.Biol.Chem. 256:10306-10312.