Hypoxanthine-guanine phosphoribosyltransferase in human erythroid cells: Degradation of the enzyme

In a previous report we provided evidence that the three major hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) isozymes in human erythroid cells are derived by posttranslational modification of a single enzyme [Johnson, G. G. et al. (1982). Biochemistry 21:960]. In the experiments reported here we provide further evidence that the modified isozymes have a catalytic activity that is at least as great as that of the unmodified enzyme. However, we also show that the total HGPRT activity decreases with red-cell age, by a factor of approximately 4, and that this decrease in activity is paralleled by a loss in HGPRT immunoreactive protein. We estimate that the loss of HGPRT activity and immunoreactive protein as well as the changes in the relative abundances of the major isozymes occur early in the cell's life.This work was supported by United States Public Health Service Grant 5 RO1 CA 16754-03 and by the San Diego State University Foundation.     

Hypoxanthine-guanine phosphoribosyltransferase: Mosaicism in the peripheral erythrocytes of a heterozygote for a normal and a mutant enzyme

A mutant form of erythrocyte hypoxanthine-guanine phosphoribosyltransferase with an abnormal isoenzyme pattern was found in a patient with a partial enzyme deficiency and X-linked gout. This abnormal pattern was a marker for the mutant enzyme in hemolysate from the heterozygote for the enzyme deficiency.These studies were generously supported by the Medical Research Council of Canada (MRC # MA4758) and the Canadian Arthritis and Rheumatism Society.     

A comparison of purine metabolism and nucleotide pools in normal and hypoxanthine-guanine phosphoribosyltransferase-deficient neuroblastoma cells

Purine nucleotide synthesis and interconversion were examined over a range of purine base and nucleoside concentrations in intact N4 and N4TG (hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient) neuroblastoma cells. Adenosine was a better nucleotide precursor than adenine, hypoxanthine or guanine at concentrations greater than 100 μM. With hypoxanthine or guanine, N4TG cells had less than 2% the rate of nucleotide synthesis of N4 cells. At substrate concentrations greater than 100 μM the rates for deamination of adenosine and phosphorolysis of guanosine exceeded those for any reaction of nucleotide synthesis. Labelled inosine and guanosine accumulated from hypoxanthine and guanine, respectively, in HGPRT-deficient cells and the nucleosides accumulated to a greater extent in N4 cells indicating dephosphorylation of newly synthesized IMP and GMP to be quantitatively significant. A deficiency of xanthine oxidase, guanine deaminase and guanosine kinase activities was found in neuroblastoma cells. Hypoxanthine was a source for both adenine and guanine nucleotides, whereas adenine or guanine were principally sources for adenine (>85%) or guanine (>90%) nucleotides, respectively. The rate of [¹⁴C]formate incorporation into ATP, GTP and nucleic acid purines was essentially equivalent for both N4 and N4TG cells. Purine nucleotide pools were also comparable in both cell lines, but the concentration of UDP-sugars was 1.5 times greater in N4TG than N4 cells.     

次黄嘌呤-鸟嘌呤磷酸核糖转移酶突变体的基因构建、表达和性质

通过对我国嗜热菌Thermoanaerobactertengcongensis中次黄嘌呤 鸟嘌呤磷酸核糖转移酶 (HGPRT)三维结构进行同源模建 ,设计出HGPRT的突变体K1 33A、K1 33S和K1 33T。用抗性筛选法 ,对HGPRT的基因进行了定点突变 ,并实现了在大肠杆菌中的高效表达。野生型HGPRT及其突变体K1 33A、K1 33S和K1 33T的催化动力学研究表明 ,HG PRT突变体改变并扩大了底物专一性 ,具有催化嘌呤类似物的活性。     

Molecular and functional analysis of hypoxanthine-guanine phosphoribosyltransferase from Arabidopsis thaliana

Hypoxanthine-guanine phosphoribosyltransferase (HGPT) occurs in both eukaryotic and prokaryotic organisms. However, the molecular and functional properties of plant HGPT are not well understood. In this study, it was found that the putative HGPT proteins from dicot and monocot plant species exhibited significant identities to their homologs from other cellular organisms. Ectopic expression of the HGPTs from Arabidopsis, soybean or wheat complemented HGPT deficiency in the hpt1 mutant of Saccharomyces cerevisiae. Recombinant Arabidopsis HGPT (AtHGPT) catalyzed both forward and reverse reactions in in vitro biochemical assays. The relative catalytic efficiency for the synthesis of guanosine monophosphate (GMP) was significantly greater than that for the production of guanine from GMP. Further investigations led to identification of the candidate residues that may form the pyrophosphate (PPi) binding loop in AtHGPT. AtHGPT expression level was dynamically regulated in Arabidopsis organs and during leaf development and senescence and seed germination. AtHGPT knockout mutant germinated more slowly than wild type control, whereas its overexpression mutant exhibited accelerated germination. Collectively, the data suggest that functional HGPTs are expressed in higher plants. In Arabidopsis, HGPT plays an active role in the salvage of purine bases and its activity is required for efficient seed germination.     

Chromosome-mediated transfer of murine alleles for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and ouabain resistance into human cell lines

Genetic drug-resistance markers were transferred via purified metaphase chromosomes from mouse L cells into the human fibrosarcoma line HT1080 and HeLa S3 cells. Interspecific chromosome-mediated transfer of hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) from mouse L cells into HGPRT^– HT1080 cells occurred at a frequency of approximately 1×10^–7. The presence of the mouse allele for HGPRT in transferent isolates was confirmed by isoelectric focusing. Transfer of ouabain resistance from mouse L cells to HT1080 and HeLa S3 cells occurred at an average frequency of approximately 4×10^–7. Expression of the mouse trait in transferent isolates was confirmed by their ability to withstand doses of ouabain which would be lethal to spontaneous ouabain-resistant mutants of the human cells but not to mouse L cells. Ouabain-resistant transferents of human cells showed 10⁴- to >10⁵-fold enhanced drug resistance, characteristic of either wild-type or mutant alleles, respectively, from ouabain-resistant donor L cells. Unstable expression of the transferred phenotypes in the absence of selection was seen in some isolates, but expression was lost at slow rates.This work was supported by National Institutes of Health Grant GM30383/21665 to RMB, Core Grants CA14051 to S. E. Luria and CA24538 to E. Mihich, and institutional predoctoral Training Grant GM07287.     

Mutagenic effect of BUdR in diploid human fibroblasts

It has only recently been possible to demonstrate the expected mutagenic effect of 5-bromodeoxyuridine (BUdR) in heteroploid hamster cells in culture. We have now extended this observation to diploid human fibroblasts utilizing techniques adapted from the work of Albertini and DeMars on X-ray mutagenesis at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in these cells. In four separate experiments, fibroblasts from a female donor were exposed to 500 micrograms/ml ethylmethane sulfonate (EMS) or 3 micrograms/ml BUdR yielding survivals of 9% and 5%, respectively. After a 6-day expression period, survivors were plated in selection medium containing 0.3 micrograms/ml 8-azaguanine (8-AG). After 3-5 weeks, azaguanine-resistant colonies were isolated for characterization or stained for counting. The average spontaneous mutation rate/cell/generation was 0.6.10(-6). The average induced mutation rates for EMS and BUdR were 7.8.10(-6) and 6.3.10(-6)/cell/generation, respectively. Similar results were obtained in two experiments with an additional fibroblast line. Mutant colonies isolated following BUdR treatment demonstrated from 1.4 to 61.5% of the HGPRT activity of the parental line and showed at least 8% Barr bodies, excluding the possibility of contamination by Lesch-Nyhan cells. This demonstration of a BUdR effect comparable to that of an alkylating agent or X-irradiation opens the study of mutation due to base-analog substitution in diploid human cells.     

Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33–258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.This work was supported by a grant from the Institute for General Medicine [GM18924 (M. W. T.)]. V. G. C. was a predoctoral fellow on Genetic Training Grant No. GM12 from the National Institute for General Medicine.     

Effect of bacitracin on erythroid differentiation of MEL cells

Bacitracin, an antibiotic widely utilized in clinical and veterinary use, was tested on murine erythroleukemia (MEL) cells. Tests were performed to evaluate the capacity of the drug to interfere with erythroid differentiation. Cells were exposed to a single treatment in S phase at sublethal doses of bacitracin. Two responses were found depending on the drug concentration. At higher concentrations (25 g/ml and 250ng/ml) a reduction in number of differentiating cells was observed but the kinetics of the process remained unchanged. At lower concentrations (from 2.5 ng/ml to 2.5 fglml) a dramatic alteration of the dynamic of differentiation was found. These two responses are related to different activities of the DNA repair mechanisms. Higher doses of bacitracin stimulate repair while lower concentrations are not able to activate repair, as demonstrated by tests with hydroxyurea. The bacitracin-induced damage can be considered a stable genetic andlor epigenetic alteration, as demonstrated by the high frequency of mutant clones isolatedfrom low-dose treated cells. The suitability of MEL cells system in evaluating genotoxicity of drugs for veterinary use is underlined.Abbreviations MEL murine erythroleukemia - HU hydroxyurea     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Cloning and restriction map of the E. coli apt gene

The apt gene coding for Escherichia coli adenine phosphoribosyl transferase has been cloned in pBR322. The restriction map of a 1.6-kb fragment containing the apt gene is presented.     

Identification of amides derived from 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained.     

Adenine phosphoribosyltransferase: A simple spectrophotometric assay and the incidence of mutation in the normal population

The significance of partial deficiency of erythrocyte adenine phosphoribosyltransferase (APRT), reported in a number of subjects with gout, has been investigated by studying its incidence in 700 normal blood donors. Three clearly deficient subjects were found, an incidence not significantly different from that in patients with abnormalities of urate metabolism. A new assay method for APRT is described in which an erythrocyte lysate is incubated with adenine and phosphoribosylpyrophosphate (PRPP) for a given time; both hemoglobin and adenine nucleotide (AMP) are then precipitated with lanthanum phosphate; the change in absorbance of adenine at 260 nm reflects the extent of its conversion to AMP by APRT.This work was supported by the National Health and Medical Research Council of Australia.     

Inhibition of MEK/ERK signalling pathway promotes erythroid differentiation and reduces HSCs engraftment in ex vivo expanded haematopoietic stem cells

The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self‐renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34⁺ cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway—PD0325901 (PD)—significantly reduces the expansion of CD34⁺ and CD34⁺ CD38^? cells, while there is no change in the expression of stemness‐related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34⁺ cells. Notably, when ERK pathway is blocked in UCB‐MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst‐forming unit‐erythroid colony (BFU‐E) as well as enhancement of erythroid glycophorin‐A marker. These results are in total conformity with up‐regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down‐regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self‐renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB‐haematopoietic progenitor cells.     

Crystal structure of a chimera of human and Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferases provides insights into oligomerization

The crystal structure of a chimera of Plasmodium falciparum (Pf) and human hypoxanthine guanine phosphoribosyltransferases (HGPRT), which consists of the core of the protein from the human enzyme and the hood region from the Pf enzyme, has been determined as a complex with the product guanosine monophosphate (GMP). The chimera can utilize hypoxanthine, guanine, and xanthine as substrates, similar to the Pf enzyme. It exists as a monomer-dimer mixture in solution, but shifts to a tetramer on addition of phosphoribosyl pyrophosphate (PRPP). The structural studies reveal that the asymmetric unit of the crystal consists of two monomers of the chimeric HGPRT. Surprisingly, the dimer interface of the chimera is the less extensive AC interface of the parent HGPRTs. An analysis of the crystal structures of the various human HGPRTs provides an explanation for the oligomeric characteristics of the chimera. Pro93 and Tyr197 form part of crucial interactions holding together the AB interface in the unliganded or GMP-bound forms of HGPRT, while Pro93 and His26 interact at the interface after binding of PRPP. Replacement of Tyr197 of human HGPRT by Ile207 in the chimera disrupts the interaction at the AB interface in the absence of PRPP. In the presence of PRPP, the interaction between Pro93 and His26 could restore the AB interface, shifting the chimeric enzyme to a tetrameric state. The structure provides valuable insights into the differences in the AB interface between Pf and human HGPRTs, which may be useful for designing selective inhibitors against the parasite enzyme.     

次黄嘌呤鸟嘌呤磷酸核糖转移酶研究进展

次黄嘌呤鸟嘌呤磷酸核糖转移酶(Hypoxanthine-guanine phosphoribosyltransferase, HPRT)是一种细胞质酶, 在体内广泛存在, 它不仅参与嘌呤碱基的补救合成途径, 而且关系到嘌呤类药物的代谢, 是调控该类药物药理效应和毒性反应的关键酶。其基因突变可影响酶的活性, 不仅可能导致不同临床表现的代谢疾病的发生, 而且影响体内嘌呤类药物的代谢。同时, HPRT作为管家基因, 是诊断许多疾病的靶点基因。文章概括了HPRT研究的新进展, 通过总结国内外研究现状, 发现HPRT的研究既推动了嘌呤类药物个体化用药的发展及新药物的研发, 又促进了HPRT突变相关遗传代谢疾病的诊断和治疗。     

Metabolic properties of an azaguanine-resistant variant of Chinese hamster ovary cells (azarts) with normal levels of hypoxanthine-guanine phosphoribosyltransferase activity

Azarts Chinese hamster ovary cells were 20 to 50 times more resistant to 8-azaguanine and 50 to 10 times more resistant to both 6-thioguanine and 6-mercaptopurine than wild-type cells. Resistance correlated with a failure of azarts cells to incorporate 8-azaguanine into the nucleotide pool and into nucleic acids. The uptake of hypoxanthine and guanine, on the other hand, was about the same in both types of cells and the hypoxanthine-guanine phosphoribosyltransferase of the azarts cells as measured in cell lysates was unaltered both in concentration and kinetic properties with hypoxanthine as well as 8-azaguanine as substrate. Plasma membrane permeability to 8-azaguanine and the regulation of intracellular pH were also not altered in azarts cells and there was no significant degradation of 8-azaguanine or azaguanine nucleotides. We conclude therefore that in azarts cells the phosphoribosylation of 8-azaguanine per se is specifically blocked but that this effect is abolished upon cell lysis.     

棉铃虫各龄期酯酶同工酶的比较

用高分辨率的薄层等电聚焦对棉铃虫各龄期幼虫的酯酶同工酶进行了比较,结果发现棉铃虫各龄幼虫都有10种相同的酯酶同工酶,它们的pl集中在pH3．5—5,5范围内。本文还从方法学的几个方面进行了探讨。     

Crystal structure of adenine phosphoribosyltransferase from Leishmania tarentolae: potential implications for APRT catalytic mechanism

The three-dimensional structure of Leishmania tarentolae adenine phosphoribosyltransferase (APRT) in complex with adenosine-5-monophosphate (AMP) and a phosphate ion has been solved. Refinement against X-ray diffraction data extending to 2.2-Å resolution led to a final crystallographic R factor of 18.3%. Structural comparisons amongst this APRT enzyme and other ‘type I’ PRTases whose structures have been determined reveal several important features of the PRTases catalytic mechanism. Based on structural superpositions and molecular interaction potential calculations, it was possible to suggest that the PRPP is the first substrate to bind, while the AMP is the last product to leave the active site, in accordance to recent kinetic studies performed with the Leishmania donovani APRT.