Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues

An enzyme sensor system has been developed to assess the freshness level in fish tissue. The system was designed to measure the K value, the concentration ratio of [Hx + HxR] and [Hx + HxR + IMP], where Hx, HxR, and IMP are hypoxanthine, inosine and inosine-5'-monophosphate, respectively. The [Hx + HxR] concentration in tissue extract was measured by nucleoside phosphorylase and xanthine oxidase immobilized on a preactivated nylon membrane and attached to the tip of a polarographic electrode. The electrode amperometrically detected the products of degradation, hydrogen peroxide and uric acid. For determination of [IMP + HxR + Hx], IMP was first converted to HxR by nucleotidase immobilized on the wall of a polystyrene tube. The enzyme electrode consisting of nucleoside phosphorylase and xanthine oxidase provided excellent reproducible results for at least 40 repeated assays and immobilized nucleotidase was good for at least 40 assays as well. The K value for each sample could be determined in ca. 10 min. When applied to K value measurements in several fish meats, the results obtained agreed well with those obtained by the conventional enzymatic method.     

齿孔酸对高尿酸血症黄嘌呤氧化酶活性的影响

采用紫外分光光度法检测齿孔酸在体外对黄嘌呤氧化酶的作用,并进行动力学研究探讨其作用机制;采用酵母联合氧嗪酸钾诱导高尿酸血症小鼠模型,观察齿孔酸对高尿酸血症小鼠血清尿酸水平、血清黄嘌呤氧化酶活性、肝脏黄嘌呤氧化酶活性及血糖血脂的影响。研究发现,齿孔酸体在外能抑制黄嘌呤氧化酶活性,降低高尿酸血症小鼠血清尿酸水平、血清黄嘌呤氧化酶活性、肝脏黄嘌呤氧化酶活性,同时明显降低空腹血糖、总胆固醇、甘油三酯、低密度脂蛋白胆固醇水平,升高高密度脂蛋白胆固醇水平,提高口服糖耐受量。结果表明,齿孔酸是黄嘌呤氧化酶竞争性抑制剂,还能缓解高尿酸血症小鼠糖脂代谢紊乱,对高尿酸血症及痛风的防治具有潜在意义。     

齿孔酸对高尿酸血症黄嘌呤氧化酶活性的影响

采用紫外分光光度法检测齿孔酸在体外对黄嘌呤氧化酶的作用,并进行动力学研究探讨其作用机制;采用酵母联合氧嗪酸钾诱导高尿酸血症小鼠模型,观察齿孔酸对高尿酸血症小鼠血清尿酸水平、血清黄嘌呤氧化酶活性、肝脏黄嘌呤氧化酶活性及血糖血脂的影响。研究发现,齿孔酸体在外能抑制黄嘌呤氧化酶活性,降低高尿酸血症小鼠血清尿酸水平、血清黄嘌呤氧化酶活性、肝脏黄嘌呤氧化酶活性,同时明显降低空腹血糖、总胆固醇、甘油三酯、低密度脂蛋白胆固醇水平,升高高密度脂蛋白胆固醇水平,提高口服糖耐受量。结果表明,齿孔酸是黄嘌呤氧化酶竞争性抑制剂,还能缓解高尿酸血症小鼠糖脂代谢紊乱,对高尿酸血症及痛风的防治具有潜在意义。     

An amperometric xanthine oxidase enzyme electrode based on hydrogen peroxide electroreduction

A xanthine oxidase enzyme electrode (xanthine oxidase immobilized on electrochemically modified graphite and conveniently coated with gelatine electrode working surface) for quantitative analysis of xanthine is proposed. The detection of thus developed electrochemical system is based on the electroreduction of hydrogen peroxide generated in enzyme layer and offered L-ascorbic and uric acid reducing interference effect on the substrate determination. At a working potential -50 mV (vs. Ag/AgCl) the detection limit of 4.5 microM and the linearity of the amperometric signal up to substrate concentration of about 40 microM were found. At that working potential, the electrode is practically inert towards L-ascorbic- and uric acid present. The response time did not exceed 2 min.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

L-阿拉伯糖对尿酸的调节作用

转录因子是一类在生物生命活动过程中起到调控作用的重要因子,参与了各种信号转导和调控过程,可以直接或间接结合在顺式作用元件上,实现调控目标基因转录效率的抑制或增强,从而使植物在应对逆境胁迫下做出反应。 WRKY转录因子在大多数植物体内都有分布,是一类进化非常保守的转录因子家族,参与植物生长发育以及响应逆境胁迫的生理过程。众多研究表明,WRKY转录因子在植物中能够应答各种生物胁迫,如细菌、病毒和真菌等;多种非生物胁迫,包括高温、冷害、高光和高盐等;以及在各种植物激素,包括茉莉酸( JA)、水杨酸( SA)、脱落酸( ABA)和赤霉素( GA)等,在其信号传递途径中都起着重要作用。 WRKY转录因子家族蛋白至少含有一段60个氨基酸左右的高度保守序列,被称为WRKY结构域,其中WRKYGQK多肽序列是最为保守的,因此而得名。该转录因子的WRKY结构域能与目标基因启动子中的顺式作用元件W￣box( TTGAC序列)特异结合,从而调节目标基因的表达,其调控基因表达主要受病原菌、虫咬、机械损伤、外界胁迫压力和信号分子的诱导。该文介绍了植物WRKY转录因子在植物应对冷害、干旱、高盐等非生物胁迫与病菌、虫害等生物胁迫反应中的重要调控功能,并总结了WRKY转录因子在调控这些逆境胁迫反应过程中的主要生理机制。     

An FIA biosensor system for the determination of phosphate.

A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at 0.7 V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-1-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100 microM phosphate, with a minimum detectable concentration of 1.25 microM phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.     

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.     

A new spectrophotometric assay for enzymes of purine metabolism. I. Determination of xanthine oxidase activity.

A new method for the determination of xanthine oxidase activity with xanthine or hypoxanthine is described. The hydrogen peroxide produced by the oxidation of the substrates is reduced by catalase in the presence of high concentrations of ethanol. The acetaldehyde formed is further oxidized by aldehyde dehydrogenase NAD or NADP-dependent. The reduction rate of the coenzymes were measured at 334 nm and utilized as indicators for the xanthine oxidase. The sensitivity of the method with xanthine as substrate can be doubled by the addition of uricase, which oxidizes uric acid to allantoin.     

Postnatal development of hepatic xanthine oxidase in baby pigs and turnover of purine catabolites at reduced ambient temperature.

1. The activity of xanthine oxidase in liver samples of baby pigs up to 4 weeks of age was investigated. On the 3rd day of life the turnover of hypoxanthine and of uric acid were measured after intravenous injection of 3H- and 14C-labelled tracers into animals kept at normal (32 degrees C) and reduced (20 degrees C) ambient temperature. 2. Hepatic xanthine oxidase activity increased progressively from 2 to 28 days of age (r = 0.689; P < 0.001). The increase of Vmax and of KM within 3-4 weeks was about 4.5-fold. 3. In 3-day-old baby pigs kept at normal temperature, pool size and turnover was about 10-fold higher for hypoxanthine than for uric acid. 4. At reduced ambient temperature, the pool size of uric acid increased 3.9-fold (P < 0.01) and turnover 1.6-fold (P < 0.05). For hypoxanthine the increases were insignificant.     

Miniaturized amperometric biosensor based on xanthine oxidase for monitoring hypoxanthine in cell culture media

Fabrication and characterization of miniaturized amperometric hypoxanthine biosensors are described and demonstrated for monitoring hypoxanthine in myocardial cell culture media. The sensors are based on xanthine oxidase (XO) immobilized on carbon fiber microelectrodes (CFMEs) using a composite film of Nafion and electropolymerized phenol (PPh). Nafion was used for XO immobilization because of its film hydrophobicity, enzyme-favored environment, and electrostatic interaction with XO, which was dispersed in Nafion film by immersing the Nafion-coated CFMEs in XO solution for 5 h. PPh film was formed as an overlay on Nafion and XO-modified CFMEs via electropolymerization. Hypoxanthine was measured with the sensor by the oxidation of enzymatic reaction products, hydrogen peroxide (H(2)O(2)), and uric acid (UA) at +0.60 V (vs Ag/AgCl). The use of Nafion and PPh as a matrix for XO immobilization yields enhanced specificity, sensitivity, and linearity toward hypoxanthine. A dynamic linear range of 5.0 microM to 1.8 mM was achieved with a calculated detection limit of 1.5 microM (S/N = 3) and a sensitivity of 3.144 nA/mM. In addition, the measurement was virtually interference-free from easily oxidizable species such as UA, ascorbic acid, physiological levels of neurotransmitters, and their principal metabolites. The biosensor was used to monitor hypoxanthine accumulation in myocardial cell culture media, in which the level of extracellular hypoxanthine was found to increase with ischemic tolerance.     

Immobilized xanthine oxidase: Kinetics, (in)stability,and stabilization by coimmobilization with superoxide dismutase and catalase

Milk xanthine oxidase was immobilized by covalent attachment to CNBr-activated Sepharose 4B and by adsorption to n-octylamine-substituted Sepharose 4B. The amounts of activity immobilized for the two preparations were 30 and 90%, respectively. The pH optima for free and adsorbed xanthine oxidase were at 8.6 and 8.2, respectively. Both free and immobilized xanthine oxidase show substrate inhibition. The apparent inhibition constant (K_i′) found for adsorbed xanthine oxidase with xanthine as substrate was higher than the K_i for the free enzyme, which was shown to be due to substrate diffusion limitation in the pores of the carrier beads (internal diffusion limitation). Higher substrate concentrations, as desirable for practical application in organic synthesis, can therefore be used with the immobilized enzyme without decreasing the rate. As a result of the internal diffusion limitation the apparent Michaelis constant (K_m′) for adsorbed xanthine oxidase was also higher than the K_m for the free enzyme. Immobilized xanthine oxidase was more stable than the free enzyme during storage at 4 and 30°C. Both forms rapidly lost activity during catalysis. The loss was proportional to the amount of substrate converted. Coimmobilization of xanthine oxidase with superoxide dismutase and catalase improved the operational stability, suggesting that O₂^? and H₂O₂ side-products of the enzymatic reaction were involved in the inactivation. Coimmobilization with albumin also had some stabilizing effect. Complete surrounding of xanthine oxidase by protein, however, by means of etrapment in a glutaraldehyde-crosslinked gelatin matrix, considerably enhanced the operational half-life. This system was less efficient than the Sepharose preparations either because much activity was lost during the immobilization procedure and/or because it had poor flow properties. Xanthine (15 mg)was converted by an adsorbed xanthine oxidase preparation and product (uric acid) was isolated in high yield (84%).     

The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate

1. The aerobic loss of GSH added to the supernatant fraction from rat liver is much increased by including the microsome fraction, which both inhibits the concurrent reduction of the GSSG formed and also augments the net oxidation rate. 2. Oxidation occurs with a mixture of dialysed supernatant and a protein-free filtrate; the latter is replaceable by hypoxanthine and the former by xanthine oxidase, whereas fractions lacking this enzyme give no oxidation. 3. In all these instances augmentation occurs with microsomes, with fractions having urate oxidase activity and with the purified enzyme; uric acid and microsomes alone also support the oxidation. 4. Evidence implicating additional protein factors is discussed. 5. It is suggested that GSH oxidation by homogenate is linked through glutathione peroxidase to the reaction of endogenous substrate with supernatant xanthine oxidase and of the uric acid formed with peroxisomal urate oxidase.     

Effect of allopurinol on the utilization of purine degradation pathway intermediates by tobacco cell cultures

Suspension cultured Nicotiana tabacum (tobacco) cells grow slowly on intermediates of the purine degradation pathway (hypoxanthine, xanthine, uric acid, allantoin, and urea) as their sole nitrogen source indicating that this degradation pathway is operative in these cells. The hypoxanthine analog, allopurinol inhibited tobacco cell growth on hypoxanthine but not uric acid. This helps confirm that the site of action of allopurinol is the conversion of hypoxanthine to uric acid by xanthine oxidase. Attempts to select cells which could grow in the presence of allopurinol with hypoxanthine as the nitrogen source were not successful.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of uricase by Candida tropicalis using n-alkane as a substrate.

Production of uricase (urate oxidase, EC 1.7.3.3) by n-alkane-utilizing Candida tropicalis pK233 was studied. Although the yeast showed very low enzyme productivity under growing conditions on glucose or an n-alkane mixture (C10 to C13) (less than 2 U/g of dry cells), enzyme formation was enhanced markedly in an induction medium consisting of potassium phosphate buffer, MgSO4, uric acid, and an n-alkane mixture (47 U/g of dry cells) or glucose (21 U/g of dry cells). Of the carbon sources tested, the n-alkane mixture was the most suitable for enzyme production. Appropriate aeration also stimulated uricase formation. In addition to uric acid, xanthine, guanine, adenine, and hypoxanthine were also effective for inducing uricase. Under optimum conditions, the maximum yield of the enzyme was 91 U/g of dry cells. Uricase thus induced was localized in the microbodies of the yeast.     

Production of uricase by Candida tropicalis using n-alkane as a substrate.

Production of uricase (urate oxidase, EC 1.7.3.3) by n-alkane-utilizing Candida tropicalis pK233 was studied. Although the yeast showed very low enzyme productivity under growing conditions on glucose or an n-alkane mixture (C10 to C13) (less than 2 U/g of dry cells), enzyme formation was enhanced markedly in an induction medium consisting of potassium phosphate buffer, MgSO4, uric acid, and an n-alkane mixture (47 U/g of dry cells) or glucose (21 U/g of dry cells). Of the carbon sources tested, the n-alkane mixture was the most suitable for enzyme production. Appropriate aeration also stimulated uricase formation. In addition to uric acid, xanthine, guanine, adenine, and hypoxanthine were also effective for inducing uricase. Under optimum conditions, the maximum yield of the enzyme was 91 U/g of dry cells. Uricase thus induced was localized in the microbodies of the yeast.     

Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions.

Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5'-nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given.     

Xanthine oxidoreductase and xanthine oxidase in human cornea

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.     

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.