A colorimetric assay for inorganic pyrophosphate that is also useful for measuring product accumulation in polymerase chain reactions

A novel coupled enzyme assay for measuring inorganic pyrophosphate (PP(i)) in biological samples is described. The total PP(i) is determined by a reaction with inosine 5'-monophosphate, catalyzed by hypoxanthine-guanine phosphoribosyl transferase, yielding hypoxanthine and phosphoribosyl pyrophosphate. The hypoxanthine is oxidized to uric acid by xanthine oxidase/xanthine dehydrogenase and can be measured by formation of formazan when a tetrazolium salt is used as the oxidant. The method is also useful for detecting and quantifying PP(i) released from nucleotides during polymerase chain reactions. This rapid and simple method for detecting amplified nucleic acids permits low-cost monitoring by eye or spectrophotometer.     

Metabolic co-operation between biochemically variant hamster cells and heterokaryons formed between these cells and chick erythrocytes.

Observations on heterokaryons formed between multiple biochemically variant polyoma virus transformed hamster cells and chick erythrocytes indicate that gain of active preformed adenosine phosphoribosyl transferase and hypoxanthine/guanine phosphoribosyl transferase immediately endows the heterokaryons with the potential to act as donors for metabolic cooperation in these systems.     

焦磷酸显色法检测PCR产物及酶活性

聚合酶链反应（polymerase chain reaction, PCR）是分子生物学领域的一项具有划时代意义的技术,但定量PCR产物或测定能生成焦磷酸的酶活性仍需要新技术的发展。本文提供了一种PCR产物定性及定量检测方法(Color PCR kit)及其用途;该方法通过焦磷酸(pyrophosphate,PPi)显色测定PCR的副产物PPi。利用试剂盒中的试剂与PPi反应,最终生成物为甲暨（formazan）,呈红色。根据显色现象(红色)判断PCR的阳性结果,由目测实现PCR的定性检测;或通过测定490 nm 处的吸光度值定量检测PCR过程中生成的副产物PPi的含量,定量检测PCR 产物的生成量;目测情况下Color PCR kit可检测到2.5 ng水平的PCR产物,用紫外分光光度计可检测到低限为1 pg;Color PCR kit法比琼脂糖凝胶电泳检测法（低限为4 ng）灵敏。Color PCR kit还可用于连接酶和转移酶活性的测定。     

Isolation and characterization of the Saccharomyces cerevisiae XPT1 gene encoding xanthine phosphoribosyl transferase

A new Saccharomyces cerevisiae gene, XPT1, was isolated as a multicopy suppressor of a hypoxanthine phosphoribosyl transferase (HPRT) defect. Disruption of XPT1 affects xanthine utilization in vivo and results in a severe reduction of xanthine phosphoribosyl transferase (XPRT) activity while HPRT is unaffected. We conclude that XPT1 encodes XPRT in yeast.     

Lack of enhanced purine biosynthesis in HGPRT- and Lesch-Nyhan cells.

The rate of de novo purine biosynthesis was measured in a series of hypoxanthine guanine phosphoribosyl transferase deficient (HGPRT-) cells from a variety of sources, including human Lesch-Nyhan cells. Under optimum growth conditions, no enhanced purine biosynthesis was detected (in contrast to previous reports). An 'elevated' level of de novo purine biosynthesis could be detected in mutants following starvation for glutamine. However, this was the result of depression of purine biosynthesis in normal cells, with a resulting artifactual overproduction in mutants.     

Selection for purine regulatory mutants in an E. coli hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant

Summary We have studied the relationship between purine salvage enzymes, 6-mercaptopurine resistance, and the purR phenotype in E. coli. Mutants resistant to 6-mercaptopurine were found to have defects in HPRT, the purR repressor, or in both. Analysis of these mutants led to the isolation of a hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant (hpt ^- gpt^-) that is extremely sensitive to adenine. Two classes of adenine resistant mutants were isolated from this strain. The first class was deficient in APRT (apt ^-) while the second class represented purine regulatory mutants (purR ^-). There is thus selection for the purR phenotype in a hpt ^- gpt^-background.Abbreviations FGAR formyl glycinamide ribotide - HPRT hypoxanthine phosphoribosyl transferase - GPRT guanine phosphoribosyl transferase - APRT adenine phosphoribosyl transferase - PRPP 5 phosphoribosyl-1 pyrophosphate - 6MP 6-mercaptopurine - FA 2-fluoroadenine     

Release of purine and pyrimidine nucleosides and their catabolites from the perfused rat hindlimb in response to noradrenaline, vasopressin, angiotensin II and sciatic-nerve stimulation.

Uric acid and uracil were released at constant rates (0.95 and 0.4 nmol/min per g respectively) by the perfused rat hindlimb. Noradrenaline, vasopressin or angiotensin II further increased the release of these substances 2-5-fold, coinciding with increases in both perfusion pressure (vasoconstriction) and O2 uptake. The hindlimb also released, but in lesser amounts, uridine, hypoxanthine, xanthine, inosine and guanosine, and all but hypoxanthine and guanosine were increased during intense vasoconstriction. Uric acid and uracil releases were increased by noradrenaline in a dose-dependent manner. However, the release of these substances did not fully correspond with the dose-dependent increase in O2 uptake and perfusion pressure, where changes in the latter occurred at lower doses of noradrenaline. Sciatic-nerve stimulation (skeletal-muscle contraction) did not increase the release of uracil, uric acid or uridine, but instead increased the release of inosine (7-fold) and hypoxanthine (2-fold). Since the UTP content as well as the UTP/ATP ratio are higher in smooth muscle than in skeletal muscle, it is proposed that release of uric acid and uracil arises from increased metabolism of the respective adenosine and uridine nucleotides during intense constriction of smooth muscle.     

Gene mutation and sister chromatid exchange in Chinese hamster cells

The mutation in hypoxanthine phosphoribosyl transferase gene and the induction of sister chromatid exchange (SCE) were comparatively studied treating Chinese hamster ovary cells with the mutagens ethylmethanesulphonate. N-methyl-N'-nitro-N-nitrosoguanidine, Mitomycin C and X-ray. All the agents exerted strong mutagenic effects and showed a dose-dependent relationship for the induction of SCEs.     

Uric Acid Metabolism in Patients with Primary Gout and the Metabolic Syndrome

Forty-four patients (40 males) with a mean age of 58 years were included in this pilot study. Mean serum urate concentration in patients with and without the metabolic syndrome (MS) was 8.8 mg/dL and 8.1 mg/dL, respectively. Urinary uric acid excretion was 543 mg/day/1.73m² in the former and 609 mg/day/1.73m² in the latter. Uric acid to creatinine ratio was 0.37 mg/mg in patients with the MS and 0.42 mg/mg in those without the MS. Mean serum urate increased from 8.6 mg/dL in subjects with three or more MS components to 10.3 mg/dL in those with five MS components. Serum urate was markedly lower in patients with mild MS (9 patients, 8.6 mg/dL) as compared to severe MS (10 patients, 9.2 mg/dL). In contrast, urinary uric acid to creatinine ratio was 0.42 mg/mg in patients with gout and mild MS and 0.33 mg/mg in gout patients with severe MS. Uric acid underexcretion appears to be more severe in gout patients with the MS. This disturbance appears to be related to the severity of the MS.     

Sexing of preimplantation mouse embryos by measurement of X-linked gene dosage in a single blastomere

Single blastomeres were isolated from zona-free 8-cell mouse embryos and assayed for X-linked hypoxanthine phosphoribosyl transferase (HPRT) activity and autosome-linked adenine phosphoribosyl transferase (APRT) activity. At this stage of development both X chromosomes are active in female embryos. Hence, a bimodal distribution of HPRT: APRT ratios, corresponding to male (XY) and female (XX) biopsied samples, was observed due to the 2-fold difference in gene dosage for HPRT activity. Batches of putative male and female embryos identified in this way were transferred to pseudopregnant recipient females. Development of the seven-eighths embryos was equivalent to that of control zona-free intact embryos. Sex determination by measurement of X-linked gene dosage was accurate and rapid enough to allow transfer of embryos of known sex without the need for cryopreservation.     

Differences between rat liver epithelial and fibroblast cells in metabolism of purines

Epithelial and fibroblast cells from adult rat liver were found to differ markedly in their metabolism of the purine hypoxanthine. Both cell types took up hypoxanthine and possessed hypoxanthine-guanine phosphoribosyl transferase for phosphoribosylating the purine. However, in the transferase assay, lysates from epithelial cells converted hypoxanthine predominantly to inosine monophosphate, with small amounts of the nucleoside inosine as product, whereas fibroblast cell lysates converted hypoxanthine predominantly to inosine. The inosine appeared not to be produced by direct ribosylation of the base, since fibroblast cell lysates had less purine nucleoside phosphorylase activity than epithelial cell lysates. Rather, the inosine produced by fibroblast lysates appeared to be derived from inosine monophosphate through catabolism of the mononucleotide by 5' nucleotidase. An inhibitor of 5' nucleotidase, thymidine triphosphate, reduced the amount of inosine formed.     

X-chromosome inactivation and enzymatic activities in diploid and triploid fibroblasts

Diploid and triploid rabbit embryos obtained by artificial fertilization were cultured. No Barr's body in XXY and only one Barr's body in XXX triploid fibroblasts was observed. Six enzymatic activities were also determined. Two X-chromosome-bound enzymatic activities, glucose-6-phosphate dehydrogenase and phosphoglycerate kinase, were significantly increased in triploid fibroblasts, while another X-chromosome-bound activity (hypoxanthine phosphoribosyl transferase) was not modified. Among the autosome-bound activities studied, pyruvate kinase and adenine phosphoribosyl transferase were not modified, whereas in the triploid cells the 6-phosphogluconate dehydrogenase activity was decreased. The relationship between these modifications of enzymatic activities in triploids and the X-chromosome inactivation is discussed.     

Introduction of a Fluorine Atom at C3 of 3-Deazauridine Shifts Its Antimetabolic Activity from Inhibition of CTP Synthetase to Inhibition of Orotidylate Decarboxylase, an Early Event in the de Novo Pyrimidine Nucleotide Biosynthesis Pathway

The antimetabolite prodrug 3-deazauridine (3DUrd) inhibits CTP synthetase upon intracellular conversion to its triphosphate, which selectively depletes the intracellular CTP pools. Introduction of a fluorine atom at C3 of 3DUrd shifts its antimetabolic action to inhibition of the orotidylate decarboxylase (ODC) activity of the UMP synthase enzyme complex that catalyzes an early event in pyrimidine nucleotide biosynthesis. This results in concomitant depletion of the intracellular UTP and CTP pools. The new prodrug (designated 3F-3DUrd) exerts its inhibitory activity because its monophosphate is not further converted intracellularly to its triphosphate derivative to a detectable extent. Combinations with hypoxanthine and adenine markedly potentiate the cytostatic activity of 3F-3DUrd. This is likely because of depletion of 5-phosphoribosyl-1-pyrophosphate (consumed in the hypoxanthine phosphoribosyl transferase/adenine phosphoribosyl transferase reaction) and subsequent slowing of the 5-phosphoribosyl-1-pyrophosphate-dependent orotate phosphoribosyl transferase reaction, which depletes orotidylate, the substrate for ODC. Further efficient anabolism by nucleotide kinases is compromised apparently because of the decrease in pK(a) brought about by the fluorine atom, which affects the ionization state of the new prodrug. The 3F-3DUrd monophosphate exhibits new inhibitory properties against a different enzyme of the pyrimidine nucleotide metabolism, namely the ODC activity of UMP synthase.     

Heterogeneity of hypoxanthine guanine phosphoribosyl transferase from human erythrocytes.

Hypoxanthine guanine phosphoribosyl transferase (E.C.: 2.4.2.8) has been purified 4000- to 4500-fold from normal human erythrocytes by three different schemes of protein fractionation. In one scheme, the enzyme was separated by preparative polyacrylamide gel electrophoresis in an LKB Uniphor system and purified by affinity column chromatography employing Sepharose/phosphoribosyl/pyrophosphate. In the second, the enzyme was isolated by isotachophoresis in the presence of Amphiline carrier ampholytes employing a Tris/phosphate/β-alanine ion system. The enzyme was then purified by isotachophoresis in the presence of carrier ampholytes using a Tris/acetate/glycine ion system. The hypoxanthine guanine phosphoribosyl transferase purified by affinity chromatography and isotachophoresis consisted, on immunoelectrophoresis, mainly of one component and had less than 5% impurities. When subjected to analytical polyacrylamide gel electrophoresis, such preparations were resolved into four isoenzymes. In the third scheme, the enzyme was isolated by isoelectric focusing. In this system, the enzyme was also resolved into four isoenzymes. Their isoelectric points were: 5.47, 5.63, 5.74, and 5.84. When subjected to analytical polyacrylamide gel electrophoresis each isoenzyme migrated at a different rate. In sodium dodecyl sulfate gel electrophoresis each isoenzyme yielded one major and one minor band. Protein appearing in the major and minor bands migrated at rates consistent with a molecular size of 33,500 and 26,500, respectively.     

Effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid and diazepam on intercellular communication in a monolayer of rat liver epithelial cells

We have studied the influence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the vitamin A derivative retinoic acid and the benzodiazepine diazepam on intercellular communication via established gap junctions in a monolayer of rat liver epithelial cells (RLB) at various times of incubation. Intercellular communication was measured as the transfer of [3H]hypoxanthine-derived nucleotides between RLB hypoxanthine guanine phosphoribosyl transferase+ (HPRT+) and RLB HPRT- cells. TPA only showed transient inhibition of metabolic cooperation: after 4 h of treatment, intercellular communication was reduced to about 40% of the control and longer treatments showed progressively less effect until 24 h of treatment, when no difference was seen between TPA-treated and control preparations. Retinoic acid was a more effective inhibitor: both 3 X 10(-6) M applied for 24 h and 10(-4) M applied for 6.5 h, caused a 50% inhibition of label transfer. The junctional communication could only be blocked at very high concentrations (5 X 10(-4) M) in short-exposure experiments, but this is possibly a consequence of non-specific effects on the cell membrane. When the incubation time was 24 h, a considerable portion of the gap junctions appeared to persist in the 'open' state. Diazepam showed no significant inhibitory effect in the experiments performed.     

Structure and sequence of mutations induced by ionizing radiation at selectable loci in Chinese hamster ovary cells

The spectrum of mutations induced by ionizing radiation at two non-essential genetic loci varies markedly. Those at the adenine phosphoribosyl transferase (aprt) locus predominantly have no detectable alterations of gene structure on Southern blots, while those at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus are largely massive deletions eliminating all coding sequence. Insertion mutations were detected at both loci. To characterize the sequence alterations producing the minor changes at the aprt locus, two mutant genes were cloned from lambda genomic libraries and sequenced. One of these mutants proved to be a 20 base-pair deletion formed between two short (3 base-pair) direct repeat sequences, while the second was the result of a 58 base-pair insertion accompanied by a 13 base-pair deletion.     

8-Azaguanine resistant african green monkey kidney cell mutans (Vero 153): isolation and characterization

Summary A clone of Vero cells resistant to up 20 μg/ml 8-azaguanine was isolated. This clone (designated Vero 153) has a doubling rate of approximately 24 h and a maximum cell density of 10,000/mm². Deficiency of the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) in Vero 153 was demonstrated by methods of radiochromatography. Vero 153 is susceptibletto hypoxanthine-thymidine-aminopterin (HAT) medium and its resistance to 8-azaguanine seems to be nonreversible. Like parenal cells, Vero 153 was also incapable of interferon production when challenged with Newcastle disease virus (NDV) or poly(inosinic acid) poly(cytidylic acid) (poly I:C). Similar chromasome complements (majority range 56 to 57) and band patterns were observed in cells harvested at Passages 10, 20, and 50. The potential use of Vero 153 for somatic cell hybridization for purposes of gene mapping, virus rescue, and the control of inteferon production is discussed. This project was supported by grant from the Medical Research Council, Canada (MT-1615).     

Human brain hypoxanthine guanine phosphoribosyltransferase: structural and functional comparison with erythrocyte hypoxanthine guanine phosphoribosyltransferase

A rapid and simple method, based on GMP Sepharose affinity chromatography, was used for the purification of human brain hypoxanthine guanine phosphoribosyltransferase. A single protein band was detected by polyacrylamide gel electrophoresis of the native purified enzyme. A subunit molecular weight of 25,000 was estimated by SDS gel electrophoresis. The Km values for hypoxanthine and phosphoribosyl pyrophosphate were 50 and 111 microM, respectively. The Ki values for GMP and IMP with phosphoribosyl pyrophosphate were 21 and 37 microM, respectively. The purified enzyme from human brain did not differ significantly from the human erythrocyte one in amino acid composition. The brain and erythrocyte hypoxanthine guanine phosphoribosyltransferases showed complete immunochemical identity on Ouchterlony double diffusion.     

Guanine salvage by organ cultures of mouse tooth germs

The effect of mycophenolic acid (MPA) which inhibits the biosynthesis of guanosine monophosphate (GMP) in organ cultures of mouse tooth germs can be partially counteracted by adding guanine to the MPA cultures. This may be due to salvaging guanine by the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), or to competition for a common membrane carrier involved in mediated transport of both guanine and hypoxanthine in normal biosynthesis and also of MPA. Experiments were carried out to compare the effect of either hypoxanthine or guanine on the MPA-caused inhibition. While addition of guanine to the MPA cultures (MPAG) supports growth equal to controls and development of dental-enamel junction (DEJ) to a level intermediate between control and MPA the addition of hypoxanthine (MPAHX) supports growth and DEJ development not better than MPA. This indicates that guanine is salvaged by HGPRT to GMP while hypoxanthine, salvaged to inosinic acid (inosinic monophosphate, IMP) is ineffective because the MPA inhibition is on the pathway from IMP to GMP.     

The gene coding a ubiquitin-activating enzyme may locate on X chromosome

tsBN75 cells which have a ts defect in the S phase have a mutation linked to the gene of hypoxanthine phosphoribosyl transferase and cannot complement ts85 cells which have a ts defect in the ubiquitin-activating enzyme. The ubiquitin-activating enzyme may be required for completion of the S phase.