Hypoxanthine Phosphoribosyltransferase Deficiency Research Articles

Purine nucleotide reutilization by human lymphoblast lines with aberrations of the inosinate cycle.

A purine nucleotide (inosinate) cycle is demonstrated with human lymphoblasts. The lymphoblast requires approximately 50 nmol of purine/10(6) cell increment. When the inosinate cycle is interrupted by the genetic, severe deficiency of either or both purine nucleoside phosphorylase (PNP) or hypoxanthine phosphoribosyltransferase (HPRT), purine accumulates in the culture medium as inosine, guanosine, deoxyinosine, and deoxyguanosine (PNP deficiency or PNP, HPRT deficiency) or hypoxanthine and guanine (HPRT deficiency). This accumulation represents an additional 25 to 32 nmol of purine which must be synthesized per 10(6) cell increment. PNP-deficient lymphoblasts have PPRibP contents characteristic of normal lymphoblasts, about 20 to 25 pmol/10(6) cells. HPRT-deficient lymphoblasts have four times higher PPRibP contents. The lymphoblast deficient for both PNP and HPRT has only a marginal elevation of PPRibP content, 1.5 times normal values. The elevated PPRibP content of HPRT-deficient cells reflects the efficient, unilateral reutilization of the ribose moiety of purine ribonucleotides and is not a cause of purine overproduction. Purine overproduction characterizing PNP-deficient lymphoblasts appears similar to overproduction from deficiency of HPRT, i.e. a break in the inosinate cycle rather than overactive de novo purine synthesis.

Studies in fibroblasts of patients with the Lesch-Nyhan syndrome and HPRT variants. Correlation of HPRT activity with hypoxanthine utilization and growth in selection media.

In the present study, hypoxanthine phosphoribosyltransferase (HPRT) has been investigated in fibroblasts of 19 patients from 16 different families with HPRT deficiency, concerning activity, incorporation of 14C-hypoxanthine, and growth in 8-azaguanine and HAT (hypoxanthine, azaserine, thymidine containing) selection media. According to these data we could classify the patients into 5 groups (patients with classical Lesch-Nyhan syndrome and patients with HPRT variants of types A, B, C, D). In 3 groups (patients with classical Lesch-Nyhan syndrome, HPRT variants C and D), a correlation of residual HPRT activity with the incorporation of 14C-hypoxanthine as well as growth in 8-azaguanine and HAT selection was observed. The variant A, from a patient with the classical Lesch-Nyhan syndrome, exhibited higher HPRT activity than that from all the other patients with the Lesch-Nyhan syndrome. However, the values of hypoxanthine incorporation and growth in selection media were as in the classical syndrome. The cells of variant B were resistant to azaguanine and grew in HAT selection media in the range of control cells, but had HPRT residual activities similar to those of variants A and C. For the characterization of the genetic heterogeneity of HPRT, it seems necessary to study the enzymatic properties in cell extracts as well as the purine uptake and proliferation of cells in different selection media.

Mechanism of purine overproduction in genetic defects of purine metabolism

A number of defects of purine metabolism are associated with hyperuricemia and/or other indicators of increased purine synthesis, whereas in another group of mutations purine production in vivo is normal. In the former group, elevated purine synthesis has been correlated with increased intracellular phosphoribosyl-pyrophosphate (PRPP) concentrations in hypoxanthine phosphoribo-syltransferase (HPRT) deficiency (Lesch-Nyhan syndrome) and PRPP synthetase overactivity, whereas in purine nucleoside phosphorylase (PNP) deficiency normal PRPP concentrations have been reported despite increased purine synthesis. We have re-evaluated PRPP metabolism in PNP-deficient fibroblasts and demonstrated that the rate of PRPP synthesis is normal but the steady-state concentration is significantly elevated. The association of increased purine synthesis with increased PRPP levels was confirmed in HPRT deficiency and PRPP synthetase overactivity, and normal values for both parameters were found in adenine phosphoribosyl-transferase and adenosine deaminase deficiencies. All the mutants studied thus conform to the rule that elevated PRPP levels are correlated with increased purine synthesis, and a causal relationship is suggested. Supported by the Academy of Finland, Sigrid Juselius Foundation, NIH (18197), and VA Medical Research Service.

Regulation of purine de novo synthesis in cultured human fibroblasts: The role of [formula omitted

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [ 14C]glycine or [ 14C]formate in α-N- formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [ 14C]formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT −) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT +). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT + cells. This inhibition was the basis of differentiation between HPRT − and HPRT + cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT − cells in culture but confirms recent studies made on human HPRT − lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate ( P- Rib-PP ), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT − and HPRT + cells. The rate of utilisation of P- Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT − cells had a 3-fold increase in P- Rib-PP content, the rate of P- Rib-PP generation was similar to HPRT + cells. Thus, in fibroblasts, the concentration of P- Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT + cells, in contrast to HPRT − cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.

Hypoxanthine phosphoribosyltransferase deficiency: association of reduced catalytic activity with reduced levels of immunologically detectable enzyme protein.

In the present study hemolysates from fourteen patients with a genetically determined deficiency of hypoxanthine phosphoribosyltransferase (EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase) activity were examined immunologically for the presence of material that crossreacts with the normal enzyme. A quantitative assay for crossreacting material in enzyme-deficient hemolysates was based on the inhibition of the immunoprecipitation of the normal enzyme. As little as 3% of normal crossreacting material could be detected. One patient in this series was found to have a normal amount of crossreacting material, whereas the remainder had no detectable crossreacting protein. The lack of detectable crossreacting material in these patients raises the possibility that a defect in synthesis or degradation of enzyme protein may be present in many patients deficient in hypoxanthine phosphoribosyltransferase.

A deficiency of hypoxanthine phosphoribosyltransferase in a sycamore callus resistant to azaguanine

Sycamore (Acer pseudoplatanus L.) calluses resistant to azaguanine were isolated after mutagenesis with nitrosoguanidine. One callus SNC61 was highly variable in its growth on 10 μg/ml azaguanine and it was concluded that its resistance was not due to a stable alteration in the callus. Another callus SNE31 was isolated after 4.5 months culture in 0.5 or 1 μg/ml azaguanine which had a 10-fold increase in resistance to azaguanine. This was shown to be accompanied by a reduction to 50% of the normal level of hypoxanthine phosphoribosyltransferase (E.C.2.4.2.8).

Incorporation of hypoxanthine by PHA-stimulated HPRT-deficient lymphocytes

Phytohemagglutinin (PHA) markedly stimulates 3H-hypoxanthine incorporation by lymphocytes of normal subjects as revealed by radioautography. There is no corresponding increase in activity of hypoxanthine phosphoribosyltransferase (HPRT) in lysates but the level of phosphoribosylpyrophosphate (PRPP), the cosubstrate for the reaction, is higher. Lymphocytes from a patient with partial HPRT deficiency responded to PHA as did the normals, whereas the response in Lesch-Nyhan (LN) subjects was variable. PHA-stimulated lymphocytes from two LN patients showed some increase in 3H-hypoxanthine incorporation, while two others failed to respond. The observations provide further evidence of genetic heterogeneity among LN patients.

Purine metabolism in normal and thioguanine-resistant neuroblastoma.

Purine metabolism has been examined in a clonal line of mouse neuroblastoma cells resistant to the cytotoxic effects of 6-thioguanine. Comparative studies in the resistant and parental lines indicate that the former cells have less than 1% of normal hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity. The activities of other enzymes important in the de novo and salvage pathways of purine biosynthesis were not significantly different in the two lines. Hypoxanthine phosphoribosyltransferase deficiency in this neuroblastoma line was associated with increased intracellular concentrations of 5-phosphoribosyl-1-pyrophosphate, an increased rate of purine biosynthesis de novo, and failure to incorporate hypoxanthine, but not adenine, into nucleotides. There are essentially the same alterations in purine metabolism that occur in hypoxanthine phosphoribosyltransferase-deficient fibroblasts or lymphoblasts derived from individuals with the Lesch-Nyhan syndrome. Clonal lines of hypoxanthine phosphoribosyltransferase-deficient neuroblastoma cells may therefore be of use in elucidating the mechanisms by which the enzyme defect leads to the neurologic dysfunction seen in children with this disease.

Disparate enzyme activity in erythocytes and leukocytes. A variant of hypoxanthine phosphoribosyl-transferase deficiency with an unstable enzyme.

A family is reported in which each of two sisters has a son with no detectable hypoxanthine phosphoribosyltransferase (HPRT) (EC 2. 4. 2. 8) in his erythrocytes, a finding considered pathognomonic of Lesch-Nyhan disease. However, neither has the stigmata of the disease. One boy is neurologically normal, and the other is moderately retarded. There was only a slight increase in urinary uric acid, but the amounts of hypoxanthine and xanthine, and their ratios, were similar to those found in Lesch-Nyhan disease, strongly indicating that excesses of these last two oxypurines are not responsible for the symptomatology in that disease. In contrast to the nondetectable HPRT activity in the red blood cells, leukocyte lysates from the two boys have 10-15% of normal activity, possibly reflecting continuing synthesis of an unstable enzyme. This hypothesis is supported by the demonstration that at 4 degrees C HPRT activity was rapidly lost in the propositus while the activity increased in control subjects. The mother's cells were intermediate between the two. The intact and disrupted leukocytes of the hemizygote, in the absence of added phosphoribosyl converted as much hypoxanthine to inosinate as the normal cell, and appropriate tests indicated that under these circumstances enzyme concentration is not rate limiting whereas the concentration of the cosubstrate, phosphoribosyl pyrophosphate, is. The capacity for normal function in the intact mutant cell is more representative of in vivo conditions than the lysate, which may explain the important modification of clinical symptomatology, the relatively mild hyperuricosuria, and the presence of mosaicism in the circulating blood cells of the heterozygotes. A similar explanation may apply to other genetic diseases in which incomplete but severe enzyme deficiencies are found in clinically normal individuals. An associated deficiency in glucose-6-phosphate dehydrogenase in this family permitted confirmation of previous observations on linkage with hypoxanthine phosphoribosyltransferase.

Adenine, hypoxanthine and guanine metabolism in fibroblasts from normal individuals and from patients with hypoxanthine phosphoribosyltransferase deficiency

The metabolism of adenine, hypoxanthine, and guanine was studied in normal fibroblasts and in fibroblasts from patients with the Lesch-Nyhan syndrome, deficient in hypoxanthine phosphoribosyltransferase activity. The cells were incubated with a radioactive purine base and the incorporation of radioactivity into all major purine nucleotides, nucleosides and bases, as well as nucleic acids, was determined. All three bases were avidly taken up and converted to nucleotides by normal cells. In the mutant cells, nucleotide synthesis from hypoxanthine and guanine was less than 2 % of normal, whereas, adenine was metabolized normally. At higher concentrations of hypoxanthine and guanine, increased utilization was demonstrated in both normal and mutant fibroblasts by direct ribosylation, probably catalyzed by nucleoside phosphorylase. The conversion of total adenine nucleotide radioactivity to guanine nucleotides was less than 2 % in the normal and less than 5 % in the mutant cells, when [ 14C]adenine was used as the substrate. Less than 10 % of total utilization of adenine and hypoxanthine was found in nucleic acids after a 6-h incubation in cells grown to confluency. The conversion of guanine to adenine nucleotides was either undetectable or below 1 % of total guanine nucleotide radioactivity. IMP was channeled mainly to adenine nucleotide synthesis. No free adenine was formed from hypoxanthine or guanine. Xanthine oxidase activity was not present in either type of cell, and inosine kinase could not be demonstrated in hypoxanthine phosphoribosyltransferase-deficient fibroblasts.