Purine metabolism in high and low uric acid lines of chickens: de novo uric acid synthesis in isolated hepatocytes and phosphoribosylpyrophosphate amidotransferase activities

The de novo biosynthesis of uric acid was examined in isolated hepatocytes from the high and low uric acid lines of chickens. Rates of incorporation of radiolabeled glycine into uric acid by hepatocytes from the high uric acid (HUA) line were approximately 3.6-fold greater than found in low uric acid (LUA) control hepatocytes. Uric acid synthesis rates in these cells were positively correlated with plasma uric acid levels (r = +0.77; P less than 0.01). The activity of phosphoribosylpyrophosphate (PRPP) amidotransferase was measured in acetone powder preparations from liver and kidney tissues of the HUA and LUA lines. Activities in kidney tissues were about 21% lower than those found in livers. PRPP amidotransferase activities in liver and kidney tissues did not correlate significantly with plasma uric acid levels. The increased synthesis of uric acid in the HUA line may be the result of the increased PRPP synthetase activities and PRPP pool sizes previously reported for these tissues.     

Phosphoribosylpyrophosphate in erythrocytes of ten mammalian species: concentration, synthesis and degradation.

1. The concentration of PRPP and the activity of PRPP synthetase have been measured in hemolysates from man and nine other mammalian species. PRPP synthetase activity was very low in dog hemolysate. 2. High concentrations of PRPP appeared to be associated with low levels of HGPRT activity, suggesting that HGPRT is the major pathway for utilization of PRPP in mammalian erythrocytes. 3. An alternative catabolic route for PRPP was observed in mammalian hemolysates, which seemed to be associated with acid phosphatase activity. The activity of acid phosphatase in mammalian hemolysates was measured.     

Alternative IMP binding in feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis

Crystal structures of Thermoanaerobacter tengcongensis hypoxanthine-guanine phosphoribosyltransferase (HGPRT) apoenzyme and the enzyme-inosine monophosphate (IMP) complex have been determined to 2.5A and 2.2A resolution, respectively. The active form of the enzyme was identified as a tetramer in solution and the K(i) value of IMP was measured to be 45 microM for alpha-D-phosphoribosyl-1-pyrophosphate (PRPP). Conformation of the flexible loop in T.tengcongensis HGPRT, which is involved in substrate PRPP binding, is different from that observed in phosphoribosyltransferases (PRTs). It contains a 3-10 helix, and a unique double serine repeat. This loop is ordered even in the apoenzyme and assumes a half-closed conformation. The primary magnesium ion is directly coordinated by side-chains of Glu101 and Asp102, and water molecules in the apoenzyme, suggesting a possible prerequisite role for substrate PRPP binding. Most interestingly, an alternative IMP binding mode is found in the structure of T.tengcongensis HGPRT-IMP complex. The 5'-phosphate of IMP occupies the PPi position usually seen in PRT-PRPP complexes. This new observation is consistent with the lower K(i) value of IMP and may suggest a mechanism involving multiple modes of interactions between IMP and T.tengcongensis HGPRT in product release and feedback inhibition. The structure of T.tengcongensis HGPRT is compared with those of mesophilic HPRTs, and several possible features contributing to its thermostability are elucidated. Overall, T.tengcongensis HGPRT appears to be more diverged from other PRTs.     

Purine biosynthesis de novo in bovine retina: purification and characterization of amidophosphoribosyl transferase and phosphoribosyl pyrophosphate synthetase.

The ability of bovine retina to synthesize purines de novo is shown for the first time. Amidophosphoribosyl transferase (EC 2.4.2.14), the enzyme controlling the rate of the process, and phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1), the enzyme regulating the intracellular contents of phosphoribosyl pyrophosphate (PRPP), were purified and characterized. The molecular masses of the enzyme subunits are similar to those of the purified enzyme from the liver. The molecular masses of amidophosphoribosyl transferase, PRPP synthetase catalytic subunit, and two PRPP synthetase-associated proteins are 50, 34, 39, and 41 kD, respectively. The apparent Km values of the enzymes and coenzymes are similar to those of the purified enzymes from the liver. For amidophosphoribosyl transferase, the apparent Km for Gln and PRPP are 0.75 +/- 0.05 and 0.66 +/- 0.09 mM, respectively (the corresponding Vmax values are 59 +/- 3 and 136 +/- 12 nmoles PPi/min per mg protein). For PRPP synthetase, the apparent Km for ribose-5-phosphate and ATP are 37.9 +/- 0.5 and 53 +/- 7 microM, respectively (the corresponding Vmax values are 61 +/- 4 and 52 +/- 3 nmoles PRPP/min per mg protein). The sensitivity of the retinal PRPP synthetase to inhibition by ADP and AMP was significantly lower than that of the enzyme from the liver.     

Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K _m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K _m of 0.033 mM), HGPRT (K _m of 0.074 mM), and PRPP amidotransferase (K _m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702.     

铁观音茶水提物对小鼠高尿酸血症的缓解作用

近年来,高尿酸血症（hyperuricemia,HUA）在人群中频发,危害性强,并发症多。为了探究日常饮品--半发酵茶铁观音茶水对于缓解HUA是否具有辅助作用,以小鼠为实验对象,采用氧嗪酸钾和次黄嘌呤联合法构建小鼠高尿酸血症模型,21只模型鼠随机分为模型组、铁观音茶水提物组、阳性药物组,7只非模型鼠作为对照。做不同处理2周后,取小鼠血清及肾、肝、小肠。观察各器官病理变化,从细胞层面鉴定铁观音茶水提物对高尿酸血症小鼠各脏器的影响;测定与HUA相关度较高的生化指标：血清尿酸（uric acid,UA）浓度、肝黄嘌呤氧化酶（xanthine oxidase,XOD）活力,以明确造模是否成功以及判断铁观音茶水提物对HUA是否具有缓解作用;利用qRT-PCR检测尿酸合成和排泄相关基因的mRNA表达水平,并利用Western blot检测肝XOD蛋白的表达水平,以从分子层面明确铁观音茶水提物对HUA的影响。病理切片显示,相比于阳性药物组,铁观音茶水提物组小鼠的肾和肝损害程度较轻;生化指标测定结果显示,铁观音茶水提物可降低血清尿酸水平,并且抑制XOD活性;从分子层面可以看出,铁观音茶水提物显著升高了尿酸重吸收转运体尿酸转运蛋白1(uric acid transporter 1,URAT1)和有机阴离子转运蛋白3(organic anion transporter 3,OAT3)的mRNA表达水平（P<0.05）,显著降低了葡萄糖转运子9（glucose transporter 9,GLUT9）和有机阴离子转运蛋白1(organic anion transporter 1,OAT1)的表达水平（P<0.05）。虽然qRT-PCR和Western blot提示,XOD的mRNA和蛋白质的表达水平升高,但尿酸生成量却下降,推测可能是铁观音茶水提物中的某种成分使得无催化活性的XOD蛋白表达增加进而对XOD基因的转录表达造成一种正反馈。研究提示,铁观音茶水提物对小鼠高尿酸血症具有缓解作用,其缓解高尿酸血症的作用与抑制XOD活性、干预尿酸生成过程和刺激或抑制相关阴离子转运体mRNA的表达相关。     

Glutamate dehydrogenase and glutamine synthetase activity in some organs of ruminants and monogastric animals.

A comparative study of glutamate dehydrogenase (GLDH 1.4.1.2) and glutamine synthetase (GS 6.3.1.2.) activity in liver, kidney and spleen homogenates from cattle, sheep, pigs and chickens showed that chicken liver contained on an average 3.5%, pig liver 8.3% and bovine liver 45.6% of the glutamate dehydrogenase activity present in sheep liver. Relatively low trace activity was found in the spleen and kidneys, except for the renal cortex of cattle (32% of activity in the liver). GS activity was the highest in chicken liver; in pigs it amounted to 33.40%, in cattle to 24.2% and in sheep to 19.7% of this activity. No marked interspecies differences were found in the values in the kidneys and spleen. It can be concluded from the results that the relatively high GLDH activity in the liver of ruminants compared with pigs and chicken is associated with the greater ability of ruminants to utilize ammonia. The higher GS activity and lower GLDH activity in chicken liver can be attributed to higher uric acid synthesis from ammonia via glutamine and purine bases and the lower ability of birds to utilize ammonia for protein synthesis. The presence of alanine dehydrogenase was not demonstrated in chicken liver, where the maximum oxidation of NADH after the addition to pyruvate and ammonia substrate was found.     

高尿酸血症与肾脏疾病关系的研究进展

高尿酸血症(HUA)是血尿酸(UA)增高的一种疾病,也是常见的机体代谢紊乱,临床上大多数HUA患者无明显的症状。由于经济水平提高和生活方式的改变,其发病率有增高趋势,且男性高于女性。HUA多发于中老年人群,严重影响其生活质量,因此中老年人群HUA已经成为普遍关注的健康问题。近年来研究发现HUA是痛风的主要病因之一,并且血尿酸(UA)水平可导致肾功能减退。已有多项研究表明HUA与肾脏疾病的发生发展密切相关,HUA是Ig A肾病、糖尿病肾病、急性肾损伤等肾脏疾病的独立危险因素,本文主要从HUA的流行病学、危险因素、发病机制及其与肾脏疾病关系这几个方面展开综述。     

Overproduction disease in man due to enzyme feedback resistance mutation. Purine overproduction in gout due to excessive activity of mutant feedback-resistant phosphoribosylpyrophosphate synthetase.

Physiologically superactive phosphoribosylpyrophosphate (PRPP) synthetase, due to feedback resistance mutation, was found in a family with excessive purine production, gout and uric acid lithiasis. The superactivity of the mutant enzyme was manifest in the propositus' erythrocytes and cultured fibroblasts, in increased generation, content and metabolic availability of PRPP, leading in the fibroblasts to acceleration of the rate of purine synthesis de novo. One of the propositus' two siblings was similarly affected, but the propositus' father, his second brother and four sons, were all clinically and biochemically normal. The mother was clinically normal and normouricemic, but hyperuricosuric. Cultured fibroblasts from her skin exhibited variability in PRPP content and availability and in the rate of purine synthesis de novo. The mother's cultures were found to contain a mosaicism of two cell populations, one with normal and the other with mutant PRPP synthetase, indicating an X-linked pattern of inheritance of the PRPP synthetase abnormality in this gouty family.     

Kidney and plasma metabolomics provide insights into the molecular mechanisms of urate nephropathy in a mouse model of hyperuricemia

Hyperuricemia (HUA) is closely associated with kidney damage and kidney diseases in humans; however, the underlying mechanisms of HUA-induced kidney diseases remain unknown. In the present study, we examined the kidney and plasma metabolic profiles in a HUA mouse model constructed by knocking out (Ko) the urate oxidase (Uox) gene. The Uox-Ko mice were characterized by an increase in uric acid, glycine, 3′-adenosine monophosphate, citrate, N-acetyl-l-glutamate, l-kynurenine, 5-hydroxyindoleacetate, xanthurenic acid, cortisol, and (?)-prostaglandin e2 together with a decrease of inosine in the kidneys. These altered metabolites confirmed disturbances of purine metabolism, amino acid biosynthesis, tryptophan metabolism, and neuroactive ligand-receptor interaction in Uox-Ko mice. Betaine and biotin were related to kidney function and identified as the potential plasma metabolic biomarker for predicting urate nephropathy (UN). Taken together, these results revealed the underlying pathogenic mechanisms of UN. Investigating these pathways might provide novel targets for the therapeutic intervention of UN and can potentially lead to new treatment strategies.     

金鱼(红龙睛)次黄嘌呤-鸟嘌呤磷酸核糖转移酶的分离及特性初步研究

 从金鱼肝脏分离并部分纯化了HGPRT。初步结果表明,该酶的分子量为78,000左右。由3个亚基组成,每个亚基的分子量约为27,000。金鱼的HGPRT很稳定,在85℃加热10分钟,至少保留80％的酶活性。该酶与PRPP的反应产物在淀粉凝胶电泳图谱上出现两条反应带。     

Impact of hand-rearing on morphology and physiology of the capercaillie (Tetrao urogallus)

Morphological and physiological disparities between 20 captive and 11 wild capercaillies were determined. Birds, their pectoral and leg muscles, hearts, livers and gizzards were weighed. The length of small intestines and caeca were measured. Haemoglobin, haematocrit, glucose, triglycerides, total protein, uric acid and thyroid hormones as well as the cytochrome c-oxidase activity of the pectoral muscle and heart were determined. The glycogen and protein contents of pectoral and leg muscles and liver were analysed. Chemical composition (water, fat, protein, ash) of muscles and liver was determined. Captive males had heavier pectoral muscles than wild ones. The result was opposite in females. Wild birds had heavier hearts, livers, and gizzards, and also longer small intestines and caeca than captive birds. The cytochrome c-oxidase activity of pectoral muscle and heart was higher in wild than in hand-reared birds. The chemical composition of livers of wild birds differed significantly from that of hand-reared capercaillies. Plasma uric acid and T(4) concentrations were higher in captive than in wild birds. The observed differences in digestive system and liver can result in diminished ability of captive birds to utilise natural food nutrients. Decreased cytochrome c-oxidase activity of hand-reared birds can affect their takeoff and flying capacity and increase their vulnerability to predation. These facts may contribute to the low survival of hand-reared birds after release.     

Studies on enzymes of nitrogen metabolism, and nitrogen end products in the developing chick (Gallus domesticus) embryo.

1. A total of 450 fertilized eggs were used to study the concentrations of uric acid, urea and ammonia in allantoic and amniotic fluids, and some enzymes of nitrogen metabolism in the liver and kidney during the development of the chick embryo from the 5th to 21st day of incubation. 2. Concentrations of the compounds studied were higher in allantoic fluid. The molar concentration of allantoic uric acid increased steadily with time. The pattern of urea and ammonia in both allantoic and amniotic fluids were the same. 3. Arginase (E.C.3.5.3.1) activity in both embryonic kidney and definitive kidney was higher than that in the liver. The specific activity of arginase (mumole urea formed/hr per g wet wt kidney) dropped during development. 4. Little arginine synthetase activity (argininosuccinate synthetase, E.C.6.3.4.5; and argininosuccinate lyase, E.C.4.3.2.1) was found in kidney, but none in the liver. 5. The complete urea cycle function was absent in both the liver and the kidney of the chick embryo.     

Elucidation of aberrant purine metabolism: application to hypoxanthine-guanine phosphoribosylstransferase- and adenosine kinase-deficient mutants, and IMP dehydrogenase- and adenosine deaminase-inhibited human lymphoblasts

We propose that the ratio of [14C]formate-labelled purine nucleosides and bases (both intra and extracellular) to nucleic acid purines provides, in exponentially growing cultures, a sensitive index for comparative studies of purine metabolism. This ratio was 4-fold greater for an HGPRT- mutant than for the parental HGPRT+ human lymphoblast line. The major components of the labelled nucleoside and base fraction were hypoxanthine and inosine. By blocking adenosine deaminase activity with coformycin we found that approx. 90% of inosine was formed directly from IMP rather than the route IMP leads to AMP leads to adenosine leads to inosine. The ratio of labelled base + nucleosides to nucleic acids was essentially unchagned for an AK- lymphoblast line and 2-fold greater than control for an HGPRT(-)-KAK- line, demonstrating that a deficiency of adenosine kinase alone has little effect on the accumulation of purine nucleosides and bases. Although adenosine was a minor component of the nucleoside and base fraction, the adenosine fraction increased from 3 to 13% with the addition of coformycin to the HGPRT(-)-AK- line. In the parental and HGPRT- lines, adenosine was shown to be primarily phosphorylated rather than deaminated at concentrations less than 5 microM. Inhibition of IMP dehydrogenase activity by mycophenolic acid caused a 12- and 3-fold increase in the rate of production of labelled base and nucleoside in the parent and HGPRT- cells respectively. These results suggest that a mutationally induced partial deficiency in the activities converting IMP to guanine nucleotides may result in an increased catabolism of IMP.     

The distinction between γ-glutamylhydroxamate synthetase and l-glutamine–hydroxylamine glutamyltransferase activities in rat tissues. Studies in vivo

The formation of gamma-glutamylhydroxamate by homogenates under optimum assay condition showed an inconstancy in the ratios of the enzyme activities utilizing l-glutamate and ATP (gamma-glutamylhydroxamate synthetase) and l-glutamine and ADP (l-glutamine-hydroxylamine glutamyltransferase) in a number of normal and neoplastic rat tissues. Although gamma-glutamylhydroxamate synthetase activities in adult livers and kidneys were identical in males and females, l-glutamine-hydroxylamine glutamyltransferase activities in the organs of females were significantly lower. The developmental formations of the two activities in liver, kidney, brain and muscle were not simultaneous. The l-glutamine-hydroxylamine glutamyltransferase activity in foetal liver or neonatal kidney could be prematurely evoked by thyroxine, but the gamma-glutamylhydroxamate synthetase activity remained unchanged. Injections of cortisol also had dissimilar effects on the two activities in thymus and hepatomas. The discrepant tissue distribution, asynchronous developmental formation and differential response to several hormonal stimuli provide evidence in vivo that the two activities are not catalysed by the same protein.     

Analysis of HGPRT- CRM+ human lymphoblast mutants.

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.     

Studies of the quaternary structure and the chemical properties of phosphoribosylpyrophosphate synthetase from Salmonella typhimurium.

Phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) was purified to virtual homogeneity from Salmonella typhimurium cells by a modification of previously published procedures. The molecular weight of the subunit was determined to be 31,000 +/- 3,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedimentation equilibrium analysis of the enzyme dissolved in 6 M guanidine hydrochloride. The amino acid composition of the enzyme was determined. Proline was identified as the only NH2-terminal residue. PRPP synthetase is apparently composed of identical or nearly identical subunits. NATIVE PRPP synthetase exists in multiple states of aggregation under all conditions. However, two predominant states were demonstrated under certain conditions. A form with molecular weight of 320,000 +/- 20,000 was found at pH 7.5 in the presence of MgATP. At pH 8.2 to 8.6, with or without MgATP, the predominant form corresponded to a molecular weight of 150,000 to 200,000; sedimentation equilibrium and velocity analysis indicated 160,000 +/- 15,000 as the most reliable molecular weight. More highly aggregated forms were observed at 4 degrees and higher protein concentrations. Removal of inorganic phosphate from PRPP synthetase by dilution or dialysis resulted in disaggregation. The fundamental unit of PRPP synthetase appears to consist of five (or possibly six) subunits, which can associate to form a dimer (10 or 12 subunits) and more highly aggregated forms. A pentameric subunit structure is consistent with the multiple species resolved by electrophoresis of the native enzyme in discontinuous polyacrylamide gel systems. Visualization of PRPP synthetase by negative staining with uranyl acetate and electron microscopy revealed fields of very asymmetric molecules, the dimensions of which corresponded to the M = 160,000 form. Dimers and higher aggregates of this unit were also seen. An unusual model, in which the five subunits are asymmetrically arranged, accounts very well for the electron microscopic appearance of the enzyme. The tendency of the enzyme to aggregate is viewed as a consequence of the unsatisfied bonding regions of the fundamental asymmetric unit.     

苯并[a]芘对小鼠肝脏和肾脏中丙二醛和过氧化氢酶的影响

目的探讨苯并[a]芘（B（a）P）对小鼠肝脏和肾脏脂质过氧化及抗氧化能力的影响。方法采用B（a）P口腔灌胃连续染毒3 d后,取肝、肾组织作匀浆,采用TBA比色法测定鼠肝脏和肾脏内的丙二醛（MDA）的含量,钼酸铵比色法测定鼠肝脏和肾脏内的过氧化氢酶（CAT）的含量。结果肝中各剂量染毒组的MDA含量增加,其中5 mg/kg、10 mg/kg剂量组与油剂对照组比较差异有显著性（P〈0.05）。肾脏中各剂量染毒组的MDA含量均有所增加,其中10 mg/kg剂量组与对照组比较差异有显著性（P〈0.05）。肝脏中各剂量染毒组的CAT的含量低剂量增加高剂量减少,肾脏中各剂量染毒组的CAT的含量增加。结论B（a）P可引起MDA含量增加诱导小鼠肝肾的脂质过氧化损伤。     

Mutagen-induced diploid human lymphoblast variants containing altered hypoxanthine guanine phosphoribosyl transferase

The human lymphoblast line MGL8 was treated with HAT and subsequently "mutagenized" with EMS (200 microgram/ml) to give 15% survival, and 6-thioguanine-resistant cells were selected by cloning in soft agarose containing the drug (1 microgram/ml). Eighteen sublines of independently derived resistant clones were isolated and studied in detail. One subline had a low residual HGPRT activity of about 1% of the parental cells. The HGPRT of this subline had a higher Km for PRPP, was more sensitive to heat, and was degraded faster by trypsin than the enzyme in extracts of MGL8 cells. This resistant subline and three others contained CRM levels of 1--38%, compared to the wild-type, so they probably represent true structural mutants of the HGPRT gene. All the variants maintained the karyotype of the parental line (46, XY, 6p-).