E. Zoref

Evidence for X-Linkage of Phosphoribosylpyrophosphate Synthetase in Man

Skin fibroblast cultures were utilized to study the mode of inheritance of a mutant feedback-resistant phosphoribosylpyrophosphate synthetase in a gouty family with purine overproduction. Selective conditions were applied to allow the survival in culture of mutant cells only. Whereas in the male gouty propositus the cell culture was homogenous for the mutant enzyme, in the cell culture from his nongouty mother two cell populations were demonstrated, one normal and the other mutant. The mosaicism in the mother is compatible with X-linkage of the enzyme. From this finding, together with the clinical and biochemical data available, it is concluded that in this family the enzyme mutation is transmitted in a X-linked recessive pattern.

Metabolic cooperation between human fibroblasts with normal and with mutant superactive phosphoribosylpyrophosphate synthetase.

METABOLIC cooperation is a form of intercellular communication by which cells in contact exchange molecules, a process providing multicellular organisms with an important mechanism for control of metabolic activity1. Subak-Sharpe et al.2 observed contact-dependent transfer of purine nucleotides from normal cells to cells mutationally incapable of producing inosinic acid due to deficiency in hypoxanthine–guanine phosphoribosyl-transferase3. Metabolic cooperation of this type was later also demonstrated with other enzymic markers, such as adenine phosphoribosyltransferase and thymidine kinase4,5. Such transfers are characterised, by the normal cell being the donor and the mutant cell being the recipient, the former transferring to the latter a mutationally lacking metabolite. We report here on a new form of contact-dependent metabolic cooperation, unique in that the transfer of a metabolite occurs from a mutant donor cell to a normal recipient cell.

Increased de novo Purine Synthesis in Cultured Skin Fibroblasts from Heterozygotes for the Lesch-Nyhan Syndrome

The metabolic consequence of hypoxanthine-guanine phosphoribosyltransferase deficiency, the accelerated rate of purine synthesis de novo, was utilized as a marker for the detection in cultured fibroblasts of heterozygosity for the Lesch-Nyhan syndrome. This marker was found to be very sensitive allowing the detection of mutant cells in nonselected mixed mutant: normal cell cultures even at low proportion of 1 to 10. Exposure of the mixed cultures to selection for the mutant cell with azaguanine increased the sensitivity of the test. Cultures from different biopsies, obtained from heterozygote females, were found to contain different proportions of the mutant cell, ranging from 10 to 84%.

Synthesis and metabolic fate of purine nucleotides in cultured fibroblasts from normal subjects and from purine overproducing mutants

Abstract The synthesis and metabolic fate of purine nucleotides were studied, employing labeled precursors, in cultured fibroblasts obtained from normal subjects and from patients with inborn excessive purine production. The patients were affected with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, with virtually complete deficiency of this enzyme and with superactivity of phosphoribosylpyrophosphate synthetase. The rate of total purine synthesis de novo was accelerated 20-fold in the hypoxanthine-guanine phosphoribosyltransferease deficient cells and by 10-fold in the cells with the phosphoribosylpyrophosphate synthetase superactivity. The proportion of the total labeling in purine nucleotides produced de novo, which was excreted into the incubation medium, was markedly increased in the cells with phosphoribosylpyrophosphate synthetase superactivity, but was similar to normal in the hypoxanthine-guanine phosphoribosyltransferase deficient cells. The labeled IMP produced de novo, or from [ 14 C]hypoxanthine, which was not degraded and excreted, was converted in both normal and mutant cells, mainly into adenine nucleotides, but in the cells with phosphoribosylpyrophosphate synthetase superactivity the conversion of labeled IMP to guanine nucleotides was significantly decreased. In all cells, [ 14 C]adenine and [ 14 C]guanine were incorporated mainly into the respective nucleotide pools, whereas the IMP formed from [ 14 C]hypoxanthine was found to be converted to adenine and guanine nucleotides in proportions similar to those after [ 14 C]formate labeling. The incorporation of adenine was increased in all mutant cells studied. The results support the hypotheses concerning the mechanism of purine overproduction in the two mutations and are in accordance with the operation in vivo of the feedback regulation mechanism at the nucleotide interconversions level. The results also suggest that in cultured fibroblasts, GMP is degraded mainly through guanosine, whereas AMP is degraded mainly through IMP.

X-Linked Pattern of Inheritance of Gout Due to Mutant Feedback-Resistant Phosphoribosylpyrophosphate Synthetase

We have recently reported a new familial enzyme abnormality associated with excessive purine production, gout and uric acid lithiasis (1–3). Phosphoribosylpyrophosphate (PRPP) synthetase in the erythrocytes and cultured skin fibroblasts from the propositus (O.G.) exhibited feedback-resistance to inhibition by several cellular compounds such as GDP and ADP. As a result the enzyme was superactive in the normal physiological milieu, and consequently PRPP content and availability for nucleotide synthesis were increased. In cultured fibroblasts the increased PRPP availability was manifest in accelerated de novo synthesis of purine nucleotides (3).

Transfer of Resistance to Selective Conditions from Fibroblasts with Mutant Feedback-Resistant Phosphoribosylpyrophosphate Synthetase to Normal Cells

A mutant phosphoribosylpyrophosphate (PRPP) synthetase, recently found in our laboratory to be the primary abnormality underlying the excessive purine production in a family affected with primary metabolic gout (1,2), was used as a marker for the study of metabolic cooperation between cultured human fibroblasts (3). In physiological cellular milieu, the mutation is manifest in superactivity of the enzyme which is due to decreased sensitivity to feedback inhibition by several physiological intracellular inhibitors such as adenosine-5′-diphosphate, guanosine-5′-diphosphate and 2,3-diphosphoglyceric acid. The superactivity of the enzyme was shown to cause increased availability of its reaction product PRPP, a key substrate for both the de novo and salvage pathways of purine nucleotide synthesis. Accordingly, the mutant cell exhibits excessive de novo synthesis of purines as well as an improved capacity to synthesize purine nucleotides by the salvage pathway. Both properties render the mutant cell a suitable marker for the study of metabolic cooperation, the increased salvage capacity allowing selection between normal and mutant cells, and the excessive de novo purine synthesis allowing determination of the proportion of mutant cells in culture containing a mixture of mutant and normal cells.

X-linked hyperuricosuria associated with uric acid lithiasis inborn erros of metabolism causing purine overproduction and uric acid lithiasis

The solubility of uric acid in both buffer and urine strongly increases in the acidic region with increasing pH (Fig. 1) (Sperling et al., 1964 and 1966). Normal urine does not contain any uric acid solubilizing substances. On the other hand, supersaturation frequently occurs in urine, especially at pH below 5.8 and is due to the presence of a nondialysable mucoprotein urine fraction (Sperling et al., 1965).

Regulation of De Novo Purine Synthesis in Human and Rat Tissue: Role of Oxidative Pentose Phosphate Pathway Activity and of Ribose-5-Phosphate and Phosphoribosylpyrophosphate Availability

5-Phosphoribosyl-1-pyrophosphate (PRPP) is a regulating substrate for the first committed and rate-limiting step in the pathway of purine nucleotide synthesis de novo. Studies in vivo and in vitro demonstrated that in human and rat tissues lowering of PRPP availability results in deceleration of purine production (1–4). On the other hand, in subjects with deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in whose tissue PRPP accumulates due to decreased consumption, and in subjects with mutant superactive PRPP synthetase who overproduce PRPP, there is an excessive production of purines (5–9).

Comment on the Assay of Purine Phosphoribosyltransferases in Cultured Human Fibroblasts

Cultured human skin fibroblasts are commonly utilized in the detection of hemizygosity and heterozygosity for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency. Two obstacles are encountered in the determination of HGPRT and adenine phosphoribosyltransferase (APRT) in extracts of cultured skin fibroblasts: the sensitivity of these enzymes in dilute cell suspension to freezing and thawing (1), and the presence of nucleotidase activity (2).