Guanine Phosphoribosyltransferase Activity Research Articles

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Hypoxanthine guanine phosphoribosyltransferase activity is related to 6-thioguanine nucleotide concentrations and thiopurine-induced leukopenia in the treatment of inflammatory bowel disease

Thiopurine drugs are widely used in the treatment of inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and adverse events occurrence. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the effects of TPMT and hypoxanthine guanine phosphoribosyltransferase (HPRT) activities on 6-thioguanine nucleotides (6-TGNs) concentrations and thiopurine-induced leukopenia in patients with IBD. Clinical data and blood samples were collected from 120 IBD patients who were receiving azathioprine (AZA)/6-mercaptopurine (6-MP) therapy. Erythrocyte TPMT, HPRT activities and 6-TGNs concentrations were determined. HPRT activity and its correlation with TPMT activity, 6-TGNs level, and leukopenia were evaluated. The HPRT activity of all patients ranged from 1.63-3.33 (2.31 ± 0.36) μmol/min per g Hb. HPRT activity was significantly higher in patients with leukopenia (27, 22.5%) than without (P < 0.001). A positive correlation between HPRT activity and 6-TGNs concentration was found in patients with leukopenia (r = 0.526, P = 0.005). Patients with HPRT activity > 2.70 μmol/min per g Hb could have an increased risk of developing leukopenia (odds ratio = 7.47, P < 0.001). No correlation was observed between TPMT activity and HPRT activity, 6-TGNs concentration, or leukopenia. High levels of HPRT activity could be a predictor of leukopenia and unsafe 6-TGN concentrations in patients undergoing AZA/6-MP therapy. This could partly explain the therapeutic response or toxicity that could not be adequately explained by the polymorphisms of TPMT.

Partial hypoxanthine–guanine phosphoribosyl transferase deficiency without elevated urinary hypoxanthine excretion

Partial hypoxanthine–guanine phosphoribosyl transferase (HGPRT) deficiency, also known as the Kelley–Seegmiller syndrome, can give rise to a wide range of neurological symptoms, and renal insufficiency. Biochemically, it is characterized by high uric acid concentrations in blood, high uric acid and hypoxanthine excretion in urine, and decreased activity of hypoxanthine–guanine phosphoribosyl transferase activity (HGPRT). However, normal uric acid concentrations in blood and uric acid excretions in urine have been reported. Here, a boy is presented with normal development and suffering from recurrent attacks of acute renal failure with slightly to clearly increased urinary uric acid excretion. Between these attacks, episodes of elevated urinary excretion of uric acid were observed with normal blood concentrations of uric acid and normal urinary excretion of hypoxanthine. HGPRT activity in erythrocytes, leukocytes, and fibroblasts was found to be strongly decreased. This case shows that not only normal blood uric acid but also normal urinary hypoxanthine concentrations do not exclude the diagnosis of partial HGPRT deficiency.

Association between Cigarette Smoking and Hypoxanthine Guanine Phosphoribosyltransferase Activity

The aim of this study was to investigate the association between smoking behavior and hypoxanthine guanine phosphoribosyltransferase (HGPRT) activity. A cross-sectional study was performed of 82 men, including 38 non-smokers and 44 smokers. Inosine monophosphate (IMP), the product of HGPRT (used as the index of activity), was measured in peripheral blood mononuclear cells using high-performance liquid chromatography. The factors potentially associated with HGPRT activity included age, glutamyl oxaloacetic transaminase, glutamyl pyruvic transaminase, cholesterol, uric acid, triglycerides, creatinine, body mass index, gout, systolic blood pressure, diastolic blood pressure, alcohol consumption, and cigarette smoking. Mean HGPRT activity was 7.05 +/- 3.44 nmol/10(6) viable cells/hour for all participants, and was significantly lower for smokers than for non-smokers (6.24 +/- 3.40 vs 7.98 +/- 3.28 nmol/10(6) viable cells/hour; p = 0.02). In addition, as the number of smoked cigarettes increased, the HGPRT activity decreased (p < 0.05). The age at onset of cigarette smoking showed a positive correlation with HGPRT activity after adjusting for smoking duration, serum uric acid, and cigarettes smoked per year using a multiple regression model (p < 0.001). We concluded that the greater the number of cigarettes smoked, the lower the HGPRT activity, and that HGPRT activity was higher in smokers who had started smoking later.

6-thioguanine resistance in a human colon carcinoma cell line with unaltered levels of hypoxanthine guanine phosphoribosyltransferase activity.

Cell populations resistant to high doses (30 microM) of 6-thioguanine (6-TG, 6-TG(r) cells) were selected from a human colon carcinoma cell line, LoVo. This cell line, which lacks hMSH2, a component of the human mismatch binding heterodimer hMutSalpha, is resistant to low doses of 6-TG. The level of activity of hypoxanthine-guanine phosphoribosyltransferase, the enzyme responsible for the phosphoribosylation of the thiopurine, was comparable to that expressed in the parental cells. No significant difference was found in the levels of enzyme activities involved in the conversion of 6-TG or its derivatives into non-toxic compounds. In contrast, a significant difference was found in the uptake kinetics of 6-TG in the 2 cell types. Net uptake of 6-TG ceased after 100-sec incubation in the 6-TG(r) cells, while it appeared to continue throughout the 10-min incubation in the wild-type cells. As a consequence, after 10-min incubation, the total amount of 6-TG taken up by the parental LoVo cells was approximately 3 times higher than that present in the 6-TG(r) cells.

Enzyme-pattern-targeted chemotherapy with tiazofurin and allopurinol in human leukemia

The hypothesis was tested that the increased IMP dehydrogenase activity in human myelocytic leukemic cells, and along with it guanylate biosynthesis, might be a sensitive target to chemotherapy by tiazofurin. 1. 1.|IMP dehydrogenase activity in normal leukocytes was 3.1 ± 0.5 (means ± S.E.)nmol/hr/mg protein and in leukemic cells it was elevated 15- to 41-fold. The activity of guanine phosphoribosyltransferase in normal leukocytes was 389 ± 27 nmol/hr/mg protein and in the leukemic cells it increased 2.8-to 6.8-fold. 2. 2.|IMP dehydrogenase was purified 4,900-fold to homogeneity from rat hepatoma 3924A with a yield of 30%. The kinetic properties of the hepatoma enzyme were similar to those of the enzyme in human myelocytic leukemic blast cells because of the similarity of the K m's for IMP (23 μ m), NAD (44 and 65 μ m); the K i for TAD was 0.1 μ m in both enzymes. 3. 3.|There was a selectivity of the in vitro response to tiazofurin in human normal and leukemic leukocytes. When labeled tiazofurin was incubated with leukocytes from normal, healthy volunteers and from leukemic patients, the leukemic leukocytes made 20- to 30-fold more TAD and the GTP content decreased as compared to normal leukocytes. This procedure proved to be a suitable predictive test in a clinical setting because patients with positive tests resonded to tiazofurin whereas those with negative ones did not. 4. 4.|The National Cancer Institute approved a chemotherapeutic phase I/II trial which concentrates on treatment of refractory acute myelocytic leukemia. Tiazofurin is infused in a 60-minute period with a pump to insure uniform delivery. A novel aspect of the trial was that it was directed primarily by the biochemical impact of tiazofurin on IMP dehydrogenase activity and GTP concentration and the tiazofurin doses were to be adjusted accordingly. Patients received allopurinol as a routine precaution against possible accumulation of uric acid in the kidney. 5. 5.|In the first eight patients, there was one complete remission, two entered the chronic phase, two entered into partial remission, one did not respond, and two were not evaluable. In the five patients who responded, there was a rapid, profound decrease in IMP dehydrogenase activity of the blast cells and a gradual decline in GTP concentrations. The blast cell count followed the decrease in the GTP concentration. The white blood cell count was largely preserved. 6. 6.|Bone marrow aspirates and peripheral blood samples showed that with tiazofurin treatment there was an induced differentiation of the myelocytes. 7. 7.|Allopurinol treatment (300 mg per kg) increased the plasma hypoxanthine concentration from the normal level of about 2 to 3 μ m to 20 to 30 μ m. In presence of low concentrations of guanine in the plasma (less than 0.1 μ m) the 10-fold elevated hypoxanthine concentration would strongly curtail the activity of the salvage enzyme, GPRT. Thus, the inhibition of blast cell IMP dehydrogenase activity by tiazofurin in presence of a markedly diminished GPRT activity caused by the allopurinol treatment resulted in a combination chemotherapeutic impact, leading to the depletion of the blast cell GTP concentration. 8. 8.|Tiazofurin treatment provided hematological remission in five patients with induced differentiation of the bone marrow. Tiazofurin therapy of refractory AML resulted in no leukopenia; therefore, this treatment avoids the morbidity and mortality encountered in conventional chemotherapy of refractory AML. 9. 9.|This clinical investigation provides evidence for the significance of the increased IMP dehydrogenase activity and the elevated capacity of the guanylate biosynthetic pathway in the expression of proliferation and differentiation of human leukocytes.

Hypoxanthine:guanine phosphoribosyltransferase activity in primary human osteosarcomas. A rationale for therapy with methotrexate-thymidine rescue?

Hypoxanthine:guanine phosphoribosyltransferase (HPRT) activity was measured in 14 human osteosarcomas to test whether a subset of these tumors was deficient in the purine salvage pathway enzyme and thus provide a rationale for therapy with methotrexate-thymidine rescue. All tumors contained HPRT activity within the range previously reported for xenografts of human osteosarcoma. Three patients received methotrexate (3.375 g/m2/24 hours) as a 72-hour continuous infusion with thymidine rescue (2.0 g/m2/24 hours) beginning 24 hours after the start of the methotrexate infusion. The methotrexate-thymidine infusion was well tolerated by all patients with no significant toxicity; however, there were no responses. We conclude that osteosarcomas are not deficient in HPRT activity. Therefore, the previously reported rationale for therapy of osteosarcoma with methotrexate-thymidine based on lack of activity of this enzyme is not valid. This combination, although well tolerated, is inconvenient and requires prolonged hospitalization. Therefore, without a valid rationale it cannot be recommended for therapy of patients with osteosarcoma.

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Schistosoma mansoni. A potential target for chemotherapy.

Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities are essential for the supply of guanine nucleotides in Schistosoma mansoni schistosomules. In crude extracts of adult S. mansoni, these two activities co-elute in size exclusion, ion exchange, and chromatofocusing chromatography and exhibit similar stabilities to heat treatment, suggesting that they are associated in one enzyme protein hypoxanthine-guanine phosphoribosyltransferase. This enzyme has been purified by a combination of heat treatment at 85 degrees C and chromatofocusing chromatography with elution at an apparent pI of 5.27 +/- 0.15. Pore gradient electrophoresis of the native enzyme followed by subsequent activity staining demonstrate an enzyme molecular weight of 105,000. The activity staining pattern remains the same whether hypoxanthine or guanine is used as the substrate, further supporting the existence of a single protein, hypoxanthine-guanine phosphoribosyltransferase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein results in a single protein band with a subunit molecular weight estimate of 64,000, suggesting that the native enzyme is a dimer. Preliminary kinetic studies showed that the purified hypoxanthine-guanine phosphoribosyltransferase reacted with guanine at a rate twice as fast as it did with hypoxanthine, but it did not act on xanthine at all. A full-length mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase cDNA clone pHPT5 and a plasmid pSV2-gpt containing the xanthine-guanine phosphoribosyltransferase gene for Escherichia coli were utilized as probes on Southern blots of S. mansoni DNA digests, and no significant hybridization was found under relatively relaxed conditions. Polyclonal antibodies made against human erythrocyte hypoxanthine-guanine phosphoribosyltransferase and E. coli xanthine-guanine phosphoribosyltransferase were tested in enzyme-linked immunosorbent assays of S. mansoni protein extracts, and no detectable cross-reacting protein was found. S. mansoni hypoxanthine-guanine phosphoribosyltransferase thus may bear rather limited homology to mammalian hypoxanthine-guanine phosphoribosyltransferase or bacterial xanthine-guanine phosphoribosyltransferase and could be an attractive target for antischistosomal chemotherapeutic drug design.

Theophylline increases serum uric acid levels

The present study was undertaken to investigate the effect of theophylline on serum uric acid and then to elucidate the mechanisms of action of theophylline as a cause of hyperuricemia. There was a significant increase of serum uric acid levels in male asthmatic patients who received theophylline compared to male control subjects without theophylline (6.3 ± 0.4 mg/ml, mean ±SEM, versus 4.3 ± 0.2 mg/ml, p < 0.01). A significant correlation of serum levels of uric acid and theophylline was demonstrated in asthmatic patients who received 200 to 400 mg sustained-release theophylline (male group, r = 0.480, p < 0.001; female group, r = 0.398, p < 0.01). Intravenous administration of aminophylline in three healthy adult male patients did not inhibit uric acid clearance, suggesting that inhibition of excretion of uric acid by theophylline is unlikely. Theophylline slightly inhibited hypoxanthine guanine phosphoribosyltransferase activity in human erythrocyte lysates at concentrations over 5 mM that is considerably more than therapeutic concentrations of theophylline as determined by the conversion of [ 14 C]hypoxanthine to [ 14 C]inosinic acid. Theophylline caused a moderate inhibition of [ 14 C]hypoxanthine uptake by K-562 cells (~50%) at 10mM that is over 100 times as high as those achieved clinically. Further studies remain to be performed to elucidate the exact mechanisms of theophylline-induced hyperuricemia.

The regulation of hypoxanthine guanine phosphoribosyl transferase activity through transfer of PRPP by metabolic cooperation

We have developed a method of relating changes in hypoxanthine guanine phosphoribosyl transferase (HGPRTase) activity to the rate of phosphoribosyl pyrophosphate (PRPP) synthesis in isolated cell lines and in co-cultures of different cell lines. Using this approach, we have determined the response of the HGPRTase activity of communication-competent and communication-incompetent cells to changes in PRPP content. The HGPRTase activity of HGPRT + communication-competent NS cells responds to changes of their own PRPP level, as well as to changes of the PRPP level of HGPRT − cells with which they are co-cultured. In contrast, the HGPRTase activity of the HGPRT +, but communication-incompetent L 929 cells responds to changes of their own PRPP content but not to changes of the PRPP content of the cocultured HGPRT − cells. These and other experiments show that PRPP is freely exchangeable between communication-competent cells and that the intracellular activity of HGPRTase in one cell can be regulated by changes in the levels of its substrate in another cell through metabolic cooperation. The results also indicate that HGPRTase normally functions at a small fraction of its total activity, and that this can be greatly increased by raising the intracellular PRPP levels. Furthermore, it is found that when communication-competent cells establish intercellular communication, they share a common pool of PRPP and of purine nucleotides. This approach can be used as the basis of a biochemical method for the quantitation of metabolic cooperation between cells.

Evidence for a genetic basis for the class A Thy-1− defect

When Thy-1− cell lines derived from different Thy-1+ murine thymic lymphomas are analyzed by complementation analysis, most fall into the “A” complementation class. A possible explanation for this result is that the Class A phenotype is due to a mutation in a gene on the X chromosome. To test this idea, selection for 6-thioguanine resistance was carried out on Thy-1+ hybrid cell lines between complementary Class A and Class C Thy-1− mutant cell lines. In some hybrid clones, there was complete concordance between 6-thioguanine resistance and a change of the phenotype of the hybrid from Thy-1+ to Thy-1−. Detailed study of one of these hybrid clones showed that 6-thioguanine resistance was accompanied by loss of hypoxanthine guanine phosphoribosyltransferase activity and that the Thy-1− phenotype was attributable to loss of the gene complementing the Class A Thy-1− mutation.

Metabolic interactions between animal cells through permeable intercellular junctions

In mixed cultures of HGPRT − (lacking hypoxanthine: guanine phosphoribosyltransferase activity) and wild-type mammalian cells coupled by permeable intercellular junctions, the purine nucleotide pools equilibrate between the two cell types prior to incorporation into nucleic acid. As a consequence, in the presence of exogenous hypoxanthine, the mutant cells stimulate the activity of HGPRT pathway in the wild-type cells and the de novo pathway of purine nucleotide biosynthesis is inhibited in both cell types. That is, the metabolic activity of a mixed culture of coupled cells is a unique characteristic of the mixture, not simply the sum of the activities of the component cells, whereas the activity of a mixed culture of cells which are not coupled by permeable intercellular junctions is simply the sum of the activities of the two cell types.

Hypoxanthine—Guanine Phosphoribosyltransferase Activity in Blood and Skeletal Muscles of Normal and Dystrophic Mice

1. Hypoxanthine--guanine phosphoribosyltransferase (HGPRT) activity was measured in erythrocyte haemolysates and quadriceps muscle extracts of normal and dystrophic 129 ReJ and C57 BL/6J mice with [8(-14)C]hypoxanthine as substrate and 5-phosphorylribose 1-pyrophosphate as a ribose 5-phosphate donor. [8(-14)C]Inosine monophosphate formed was separated by high-voltage electrophoresis and radioactivity was measured by liquid-scintillation counting. 2. In erythrocyte haemolysates, HGPRT activity was similar in normal and dystrophic C57 BL/6J mice but was significantly higher in dystrophic than in normal 129 ReJ mice. Elevated enzyme activity was observed only in mice that were clinically severely affected. 3. In muscle homogenates, HGPRT activity was significantly higher in dystrophic than in normal animals of both 129 ReJ and C57 BL/6J mice. Enzyme activity was not related to the severity of the disease. 4. It is suggested that changes in erythrocytes are secondary to the dystrophic process and that elevated HGPRT activity in skeletal muscle may be related to abnormal energy metabolism, possibly via the pentose monophosphate shunt.

Study of the role of puring phosphoribosyltransferases in the uptake of adenine and guanine by Schizosaccharomyces pombe cells.

1. In the yeast Schizosaccharomyces pombe 972h-, the uptake rate of both adenine and guanine is related to variations in the specific activity of the corresponding phosphoribosyltransferases during the growth of the culture. Furthermore, the mutant strains dap 1, devoid of adenine phosphoribosyltransferase activity, and pur 1, devoid of guanine phosphoribosyltransferase activity have a lowered uptake rate of adenine and guanine respectively, along with an increased apparent Km value for these purines in comparison to the wild-type 972h. 2. The uptake rate of the purines is strongly dependent on the pH of the uptake medium in 972h- as well as in the strains dap 1 and pur 1, the optimum being between pH4 and pH5. 3. A new method of extraction of 5-phosphoribosyl-1-pyrophosphate from the yeast has been devised. Important fluctuations of the P-Rib-P2 pool were measured in S. pombe at different stages of growth, the maximum taking place at the start of the exponential phase, whereas no variations in the specific activity of the P-Rib-P2 synthetase could be observed during the growth. The P-Rib-P2 intracellular content in the mutants devoid of purine phosphoribosyltransferases, namely pur 1, dap 1 and pur 1, dap 1, was increased up to 5-fold as compared to the wild-type strain. 4. The effect of intracellular concentrations of P-Rib-P2, a substrate for phosphoribosyltransferases, on the uptake rate of purines has been studied: addition of formycin to the growth medium lowered simultaneously the P-Rib-P2 intracellular content and the uptake of adenine and guanine. 5. Although our results demonstrate the activating effect of phosphoribosyltransferase activities on the uptake of adenine and guanine, they do not support the hypothesis of a 'group translocation' mechanism.

6-Azauridine, an inhibitor of the purine salvage pathway.

Since the time when the knowledge about nucleic acid metabolism has been extended and the connection to malignant processes was recognized efforts were made to find new inhibitors interfering with different stages of nucleic acid metabolism. One of the pathways involved in these processes is the purine salvage pathway. Henderson (1) and Lau (2) examined different purine and pyrimidine derivatives and analogues for their ability to inhibit the enzymes of the purine-phosphoribosyltransferase. Henderson (1) found 13 compounds out of 116 chemicals which showed more than 75% inhibition of the adenine-phosphoribosyltransferase. Most of them were adenine analogues. Lau (2) on the other hand, investigated different purine and pyrimidine derivatives to inhibit the hypoxanthine- guanine-phosphoribosyltransferase activity of human erythrocytes. There were only some purine and purine nucleoside analogues active inhibitors of these enzymes. Similar experiments were reported by Lau (3) of Ehrlich ascites tumor cells.

Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.

Appearance of hypoxanthine guanine phosphoribosyltransferase activity as a consequence of mycoplasma contamination.

MYCOPLASMAS are common contaminants of cells in culture and are often responsible for profound alterations in the metabolism of infected cells1,2. Such alterations include chromosomal aberrations3,4 and changes in host cell enzyme activities5. We have reported that amniotic fluid cell cultures contaminated with mycoplasmas revealed a significant increase in chromosomal aberrations over those seen in uninfected cultures6. This finding demonstrated that the presence of these organisms in amniotic fluid cell lines poses a potential hazard to prenatal chromosome diagnosis.

Use of Escherichia coli mutants to evaluate purines, purine nucleosides, and analogues.

Of 142 purines, purine nucleosides, and analogues tested for inhibition of growth of Escherichia coli B Hill, 45 were active. Of these, 27 were evaluated for inhibition of other E. coli lines, including those resistant to 6-thioguanine, 2-fluoroadenosine, 2,6-diaminopurine, or 6-mercaptopurine. Most toxic to the parent lines were 2-fluoroadenosine, 2-fluoroadenine, 2-fluoro-5'-deoxyadenosine, adenosine, 6-thioguanosine, 6-thioguanine, 6-mercaptopurine, 6-mercaptopurine ribonucleoside, 2-azaadenine, 2'-deoxyinosine, 6-N-aminoadenine, and inosine. Hypoxanthine was strongly inhibitory only to E. coli B Hill. Evidence regarding the substrate specificity of the three purine phosphoribosyltransferases was obtained by assaying for these enzymes in extracts of the various cell lines and by cross-resistance studies. The line selected for resistance to 6-thioguanine had low guanine phosphoribosyltransferase activity (guanosine monophosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and was deficient in activity for xanthine and 6-thioguanine. The lines selected for resistance to 2-fluoroadenosine and 2,6-diaminopurine were deficient in adenine phosphoribosyltransferase activity (adenosine monophosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7), and that selected for resistance to 6-mercaptopurine had low hypoxanthine phosphoribosyltransferase activity and undetectable activity with 6-mercaptopurine as a substrate. Purine, 6-methylpurine, 2-fluoroadenine, 2,6-diaminopurine, and 2-azaadenine were classified as adenine analogues; 6-mercaptopurine and 8-aza-2,6-diaminopurine, as hypoxanthine analogues; and 6-thioguanine and 2-amino-6-chloropurine, as analogues of guanine. The inhibition of bacterial growth by hypoxanthine, inosine, 2'-deoxyinosine, or adenosine was prevented by small amounts of thiamine or by relatively high concentrations of either cytidine or uridine. Cytidine also reversed the inhibition by some purine and purine ribonucleoside analogues. Orotate phosphoribosyltransferase (OMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.10), a possible site of action for these compounds, was not inhibited directly by the toxic agents.

Reappearance of murine hypoxanthine guanine phosphoribosyl transferase activity in mouse A9 cells after attempted hybridisation with human cell lines

A method for the electrophoresis of hypoxanthine guanine phosphoribosyl tranferase (HGPRT) on starch gels is described which can distinguish human and murine HGPRT and resolve each into a number of bands. Five putative hybrid lines, made between HGPRT-deficient (HGPRT −) mouse fibroblasts (A9 cells) and HGPRT + human cells (lymphocytes, fibroblast and lymphoblast cell lines), and which were able to grow in the presence of HAT, were tested for their HGPRT type. The expected human HGPRT was seen in only two of the five lines and in one of these murine HGPRT was also present. The line with the mixture of HGPRT types lost the human enzyme on further subculture. The remaining three lines had only murine HGPRT. The reversion of HGPRT- to HGPRT + in A9 cells is an extremely rare event and the presence of murine HGPRT in four of the five cell lines studied, and in none of the control A9 cultures, indicates a very high rate of reversion (derepression) in fusion experiments with human cells. Where human HGPRT was detected, both human and murine glucose-6-phosphate dehydrogenase, together with a hybrid band, was seen, and also a mixture of human and murine phosphoglycerate kinase with no hybrid band. Human X chromosomes were cytologically demonstrable where the three human enzymes were present and in later subcultures of the line with no murine HGPRT, human PGK became lost whereas the two other human X-linked enzymes were consistently found.

Effects of glutamine on purine metabolism in the lesch-Nyhan syndrome

Glutamine (GLN) is the amino donor in two reactions of de novo purine synthesis and in the formation of guanylic from xanthylic acid (XMP). When confluent cultures of fibroblasts from normal individuals or from patients with the Lesch-Nyhan syndrome (LN) are maintained in GLN-free medium for 24 hours, their rate of de novo synthesis decreases to one-tenth of control cultures. Addition of GLN causes a 5–15-fold stimulation in both types of cells, but at comparable levels of GLN the rate in LN fibroblasts remains 2–5 times higher than normal. In GLN-free medium, LN cells convert 50% more adenine-14C to nucleotides than do normal cells. Of the radioactivity taken up, 1.1% is diverted to guanine nucleotide synthesis in the normals, and 2.8% in LN fibroblasts (sum of radioactivity in guanine nucleotides, XMP, the corresponding nucleosides and bases). In GLN-free medium, XMP, xanthosine, and xanthine contain 67% of this total in the mutants and 43% in the normals. Addition of GLN brings about a decrease in the total incorporation into the componets of the pathway, but a net increase in guanine nucleotide synthesis, as only 4% of the radioactivity is now found in the xanthine compounds in the mutants and 8.8% in the normals. The results show that the availability of GLN can be rate-limiting not only to de novo purine synthesis, but also to guanine nucleotide formation at the XMP aminase reaction. LN cells appear to be more sensitive to GLN deprivation in this reaction, and yet are solely dependent on it for guanine nucleotide synthesis, as they are grossly deficient in hypoxanthine guanine phosphoribosyltransferase activity.