Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.

Activity of sequential low-dose methotrexate and fluorouracil in advanced colorectal carcinoma: attempt at correlation with tissue and blood levels of phosphoribosylpyrophosphate.

Forty-five patients with metastatic colorectal carcinoma were treated with low-dose methotrexate (MTX) and 5-fluorouracil (5-FU) given sequentially. The dose of MTX was 40 mg/m2 intravenously (IV) on days 1 and 8 followed 24 hours later by 5-FU at 600 mg/m2 IV on days 2 and 9; the drugs were recycled every 28 days. Fourteen (32%) of 43 adequately treated patients had a complete or partial response lasting a median of nine months (range, 6-15 + months). Four patients had a minor response and seven patients had stable disease for a median of nine and 10 months, respectively. Toxicity included mucositis in 28 (65%) patients, diarrhea in 18 (40%), nausea in 11 (24%), and vomiting in seven (16%). Hematologic toxicity was mild: six patients had nadir white blood cell counts less than 3.5 X 10(3) cells/microL, and seven patients had a nadir platelet count less than 100 X 10(3) cells/microL. Serial biopsies and blood samples were obtained in selected patients to evaluate the effect of MTX on tissue and lymphocyte phosphoribosylpyrophosphate (PRPP) and PRPP synthetase levels.

Acquired increases of human erythrocyte purine enzymes.

The alterations of three erythrocyte purine enzymes were studied in 12 patients with diseases associated with reticulocytosis, two patients with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, seven patients with severe megaloblastic anemia, and 14 normal subjects. The specific activity of adenine phosphoribosyltransferase was positively correlated (r = 0.81) with the reticulocyte percentate in ten patients with a normal hypoxanthine-guanine phosphoribosyltransferase. Two apparent types of alterations of this enzyme were distinguished: (1) increased specific activity with a normal half life as in megaloblastic anemia, and (2) a prolonged half life with or without an elevation of specific activity as in the deficiency of hypoxanthine-guanine phosphoribosyltransferase. Hypoxanthine-guanine phosphoribosyltransferase and phosphoribosylpyrophosphate synthetase were increased in megaloblastic anemia, but were not correlated with the reticulocyte percentage and did not have a consistent change in the half life in the other disorders studied. The data show that acquired disorders associated with reticulocytosis may cause an elevation of the specific activity of purine enzymes in peripheral circulating erythrocytes. Therefore, these factors must be carefully considered in the interpretation of an elevated level of enzyme activity.

Normal activity of metabolic pathways involved in the formation and utilization of phosphoribosylpyrophosphate in erythrocytes of patients with primary metabolic gout.

The activity of metabolic pathways involved in the formation and utilization of phosphoribosylpyrophosphate (PRPP) was studied in. The erythrocytes of 34 patients with idiopathic metabolic gout. The activities of the oxidative pentose shunt, of the hypoxanthine-guanine and adenine phosphoribosyltransferases (HGPRT, APRT) and of PRPP synthetase, as well as the rates of PRPP generation and of adenine incorporation into nucleotides were found to be normal in the erythrocytes of all these patients. Four patients with metabolic gout due to enzymatic abnormalities, two relatives with partial deficiency of HGPRT and two relatives with mutant feedback-resistant PRPP synthetase, were studied for comparison. The significance of the results is discussed in relation to postulated mechanisms for purine overproduction in metabolic gout.

Phosphoribosylpyrophosphate synthetase in human erythrocytes: assay and kinetic studies using high-performance liquid chromatography.

A method using high-performance liquid chromatography (HPLC) for determination of phosphoribosylpyrophosphate (PRPP) synthetase activity in human erythrocytes has been developed and PRPP synthetase activity on purine and pyrimidine metabolic disorders has been studied. Kinetic properties of erythrocyte PRPP synthetase of patients with gout and of a patient with pyrimidine 5'-nucleotidase deficiency were compared with those of healthy subjects. The mean of PRPP synthetase activity of gouty patients was a little higher (P less than 0.01) than that of healthy subjects. The response of the enzyme for ATP of gouty patients was different from that of healthy subjects. The shapes of activation curve of the enzyme for inorganic phosphate were hyperbolic in gouty patients and in a patient with pyrimidine 5'-nucleotidase deficiency.

Phosphoribosyl pyrophosphate synthetase and glutathione reductase in erythrocytes from hyperuricaemic and gout patients.

The enzymic activities of erythrocyte phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and glutathione reductase (EC 1.6.4.2) have been measured in 54 primary gout patients, 35 individuals having hyperuricaemia and 51 healthy controls. Statistical analyses have shown a significant increase (p less than 0.01) in the enzymic activity of erythrocyte PRPP synthetase in both the hyperuricaemic and gout groups compared with the controls. No correlation between activity and age was found in any of the three clinical groups. A significant decrease (p congruent to 0.01) was found in the enzymic activity of red cell glutathione reductase in the gout group compared with the other two groups. The biochemical significance of the changes in enzymic activities of the two enzymes in primary gout is discussed.

A simple and sensitive method for estimating the concentration and synthesis of 5-phosphoribosyl 1-pyrophosphate in red blood cells.

A method is presented for the determination of 5-phosphoribosyl 1-pyrophosphate (PRPP), which is based on the release of 14CO2 from [carboxyl-14C]-orotic acid by the consecutive action of orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. The assay is simpler and less time-consuming than most methods currently employed and is equally sensitive. The method proved to be suitable for measuring low concentrations of PRPP such as found in human erythrocytes and fibroblasts. An increased PRPP concentration was observed in erythrocytes from patients with partial or complete deficiency of hypoxanthine-guanine phospho-ribosyltransferase. frp, sp,e (but not all) gouty patients and from a patient with deficiency of purine nucleoside phosphorylase. PRPP synthetase activity was measured with a method similar to the assay for PRPP. In erythrocytes with an increased PRPP concentration, PRPP synthetase activity was found to be normal at both optimal and suboptimal substrate concentrations.

An HPLC-linked assay of phosphoribosylpyrophosphate synthetase activity in the erythrocytes of adults and children with neurological disorders.

A two-step non-radioactive method that uses reverse-phase high-performance liquid chromatography (RP-HPLC) is described for the determination of phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) activity in human erythrocytes. The method is accurate and easily reproducible in different chromatographic systems; it is based on the quantification of phosphoribosylpyrophosphate by conversion into orotidine monophosphate and uridine monophosphate. Phosphoribosylpyrophosphate synthetase activity was determined in the erythrocytes of healthy adults and children, the latter showing significantly higher activity than the former. The enzyme activity assayed in children with different neurological disorders was significantly lower in patients with Rett syndrome than in control children or in autistic or mentally retarded patients.

A simplified method for the determination of phosphoribosylpyrophosphate synthetase activity in hemolysates.

Methods currently employed for measurement of phosphoribosylpyrophosphate synthetase (ribophosphate pyrophosphokinase; PRPPs) activity are cumbersome and expensive, requiring an auxiliary enzyme reaction, a radioactive nucleobase and multiple incubations, or do not allow a complete kinetic study. The aim of the present study is to describe a simplified, single step, non-isotopic method for determination of PRPPs in hemolysate, appropriate for a complete screening of PRPPs activity disorders. Briefly, the charcoal-treated hemolysate is incubated with saturating amounts of substrates and P1 P5 di(adenosine 5') pentaphosphate (Ap5A), an inhibitor of human adenylate kinase, to prevent conversion of AMP to ADP. AMP generated in this reaction is then measured by HPLC. Adenylate kinase activity was fully inhibited by Ap5A, allowing the accurate determination of AMP. The method was sensitive and reproducible and mean and variance values compared closely with those reported using other, more complicated, assay procedures. The hyperbolic curve relating Pi concentration to initial reaction velocity was shifted to sigmoidal by addition of 0.02 mmol/l GDP which inhibited PRPPs activities only at inorganic phosphate concentrations < 2 mmol/l. This suggests that this method should provide sensitive and accurate screening for regulatory, as well as catalytic, defects underlying PRPPs superactivity.

A spectrophotometric assay of phosphoribosyl pyrophosphate synthetase.

A simple spectrophotometric assay of phosphoribosyl pyrophosphate synthetase from human erythrocytes is described. The kinetic properties of the enzyme have been determined and are in good agreement with published results. Conditions for storing haemolysates at -20 degrees C are described. The method is suitable for semi-automation.

[Isoenzymes of blast cells in malignant hemopathies: present state and prospectives]. / Isoenzymes des cellules blastiques au cours des hémopathies malignes: actualité et prospective.

Numerous isoenzymes are used as markers in the course of malignant haemopathies. These are notably the isoenzymes of lactic dehydrogenase, hexosaminidase, esterases, acid phosphatases, and thymidine kinase. Their study already permits a finer and more rigorous classification of leukaemias and makes it possible to entertain serious hopes in at least three spheres: a better choice of treatment, surveillance of therapeutic efficacy and of remissions, and the development of new modes of therapeutic action by means of selective inhibitors.

[Clinical and biochemical heterogeneity of the Lesch-Nyhan syndrome]. / Klinicheskaia i biokhimicheskaia geterogennost' sindroma Lesh--Nikhana.

The screening program directed towards a detection of patients with hereditary enzymopathy allowed to reveal 8 boys from 4-14 years with the Lesch-Nyhana syndrome. Among these children 4 were studied in detail clinically and biochemically. The study demonstrated that there were different degrees of mental retardation, autoagressive or agressive behaviour. The activity of erythrocyte hypoxanthinguanine phosphoribosyltransferase (HGPRT) in 2 cases was not changed, in 1-decreased and in 1--was absent. In 1 case of a decreased activity of hypoxanthinguanine phosphoribosyltransferase there was a drastic drop in the activity of adeninphosphoribosyltransferase (APRT). The study showed that there was a double increase in the cerruloplasmin activity in the blood plasma and of the cytochromoxydase in the leukocyte mitochondria. This indicates that the genetically determined drop in the HGPRT or APRT leads to disturbances in the other links of metabolism in the organism. The clinical manifestation of the disease due to a disturbed metabolism of biogen amines in the CNS is postulated.

[Phosphoribosyl pyrophosphate and its metabolic enzymes in the erythrocytes in certain forms of anemia]. / Fosforibozilpirofosfat i fermenty ego obmena v éritrotsitakh pri nekotorykh formakh anemii

Content of phosphoribosyl pyrophosphate and the activity of ribose phosphate pyrophosphokinase and AMP-pyrophosphorylase were studied in erythrocytes of healthy persons and of patients with various types of anemia. Deficiency of phosphoribosyl pyrophosphate and a decrease in the activity of enzymes studied were observed in erythrocytes under microspherocytic and hypoplastic anemia. The alterations correlated with impairments in energy metabolism. At the same time an activation of phosphoribosyl pyrophosphate enzymatic system and an increase of its content in erythrocytes did not depend on energy metabolism in Marchiafava--Micheli disease.

Hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiencies: HPRT1 mutations in new Japanese families and PRPP concentration.

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout with hyperuricemia. Four mutations were detected in two Lesch-Nyhan families and two families with partial deficiency since our last report. A new mutation of G to TT (c.456delGinsTT) resulting in a frameshift (p.Q152Hfs*3) in exon 3 has been identified in one Lesch-Nyhan family. In the other Lesch-Nyhan family, a new point mutation in intron 7 (c.532+5G>T) causing splicing error (exon 7 excluded, p.L163Cfs*4) was detected. In the two partial deficiency cases with hyperuricemia, two missense mutations of p.D20V (c.59A>T) and p.H60R (c.179A>G) were found. An increase of erythrocyte PRPP concentration was observed in the respective phenotypes and seems to be correlated with disease severity.