• Journal of Microbiology and Biotechnology
• /
• v.25 no.6
• /
• pp.887-892
• /
• 2015

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuDof P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.

• Journal of Plant Biology
• /
• v.36 no.4
• /
• pp.415-423
• /
• 1993

대두의 uricase II cDNA를 탐침으로 plaque 혼성화 방법에 의해 해녀콩의 뿌리를 cDNA library로부터의 두 개의 phage 클론(λCINUO-01, λCINUO-02)을 선별하였다. 두 phage 클론은 약 1.6 kb와 1.0 kb의 insert를 갖고 있었으며 이들의 염기서열을 결정하기 위하여 pUC19과 pBSKS vector에 subcloing(pcCLNUO-01, pcCLNUO-02)하였다. Sanger법에 의해 염기서열을 결정한 결과, 두 클론은 각각 1,611 bp와 1,024 bp로 이루어져 있었으며 pcCINUO-01은 308개의 아미노산, pcCINUO-02는 301개의 아미노산을 암호화하는 open reading frame(ORF)을 갖고 있었다. 두 클론의 ORF의 염기서열은 대두의 uricase II와 각각 88.9%, 89.3%의 상동성을 보여주었으며, 아미노산 서열은 84.1%, 85.4%의 상동성을 보여주었다. pcCINUO-01의 경우, 종결코돈으로부터 313 NT 하류쪽에 진핵생물의 poly(A) 첨가신호인 AATAAA 서열이 존재하였으며 이로부터 21 NT 하류쪽에 17 잔기의 poly(A)가 존재하였다. 두 클론의 염기서열에서 추정된 아미노산 서열의 카르복시 말단에는 세포질에서 합성된 몇몇 단백질들이 peroxisome으로 수송되는데 필요한 신호서열인 Ser-Lys-Leu-COOH 서열이 존재하고 있었다. 두 클론의 염기서열을 토대로 아미노산 조성을 살펴본 결과, 염기성 아미노산(Arg, His, Lys)과 산성 아미노산(Asp, Glu)이 각각 46 대 35, 47 대 35의 비를 보여주었는데 이는 uricase II 단백질의 염기성 성질을 보여주는 결과로 추정된다. Northern 혼성화 결과 해녀콩에서 uricase II는 뿌리혹에서만 특이적으로 발현됨을 알 수 있었고 게놈 혼성화 반응 결과는 uricase II 유전자가 해녀콩 게놈상에 유전자 가족으로는 존재할 수 있음을 보여주었다.

• Journal of Plant Biology
• /
• v.28 no.3
• /
• pp.225-232
• /
• 1985

Intracellular localization and the developmental changes in activities of uricase and allantoinase during germination were investigated with the cotyledons of rape(Brassica napus L.) seedlings. The development anddisappearance of uricase activity took place independently of light, but allantoinase activity was increased by light. The temporal pattern of uricase activity showed that uricolysis was actively taking place in the cotyledons during their early stages of germination. While uricase can be localized in the microbody fraction isolated from crude organelle extracts of the cotyledons by density gradient centrifugation, most of the allantoinase activity found in the microbody fraction did not appear to be an integral part of the microbody.

• Molecules and Cells
• /
• v.22 no.2
• /
• pp.141-145
• /
• 2006

Uric acid is the end product of the purine degradation pathway in humans. It is catabolized to allantoin by urate oxidase or uricase (E.C. 1.7.3.3.) in most vertebrates except humans, some primates, birds, and certain species of reptiles. Here we provide evidence that mouse transthyretin-related protein facilitates the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. Mutagenesis experiments showed that the residues that are absolutely conserved across the TRP family, including His11, Arg51, His102, and the C-terminal Tyr-Arg-Gly-Ser, may constitute the active site of mTRP. Based on these results, we propose that the transthyretin-related proteins present in diverse organisms are not functionally related to transthyretin but actually function as hydroxyisourate hydrolases.

• Molecules and Cells
• /
• v.38 no.2
• /
• pp.187-194
• /
• 2015

Thioredoxin (TRX) is a disulfide reductase present ubiquitously in all taxa and plays an important role as a regulator of cellular redox state. Recently, a redox-independent, chaperone function has also been reported for some thioredoxins. We previously identified nodulin-35, the subunit of soybean uricase, as an interacting target of a cytosolic soybean thioredoxin, GmTRX. Here we report the further characterization of the interaction, which turns out to be independent of the disulfide reductase function and results in the co-localization of GmTRX and nodulin-35 in peroxisomes, suggesting a possible function of GmTRX in peroxisomes. In addition, the chaperone function of GmTRX was demonstrated in in vitro molecular chaperone activity assays including the thermal denaturation assay and malate dehydrogenase aggregation assay. Our results demonstrate that the target of GmTRX is not only confined to the nodulin-35, but many other peroxisomal proteins, including catalase (AtCAT), transthyretin-like protein 1 (AtTTL1), and acyl-coenzyme A oxidase 4 (AtACX4), also interact with the GmTRX. Together with an increased uricase activity of nodulin-35 and reduced ROS accumulation observed in the presence of GmTRX in our results, especially under heat shock and oxidative stress conditions, it appears that GmTRX represents a novel thioredoxin that is co-localized to the peroxisomes, possibly providing functional integrity to peroxisomal proteins.

• Journal of the Korean Electrochemical Society
• /
• v.19 no.2
• /
• pp.29-38
• /
• 2016

Screen printed carbon electrodes (SPCEs) with immobilized osmium-based hydrogel redox polymer, uricase and PEGDGE can be used to apply uric acid electrochemical detecting. The osmium redox complexes were synthesized by the coordinating pyridine group having different functional group at 4-position with osmium compounds. The synthesized poly-osmium hydrogel complexes are described as PAA-PVI-$[Os(dCl-bpy)_2Cl]^{+/2+}$, PAA-PVI-$[Os(dme-bpy)_2Cl]^{+/2+}$, PAA-PVI-$[Os(dmo-bpy)_2Cl]^{+/2+}$. The different concentrations of uric acid were measured by cyclic voltammetry technique using enzyme-immobilized SPCEs. The prepared SPCEs using PAA-PVI-$[Os(dme-bpy)_2Cl]^{+/2+}$ showed no interference from common physiologic interferents such as ascorbic acid (AA) or glucose. The resulting electrical currents at 0.33 V vs. Ag/AgCl displayed a good linear response with uric acid concentrations from 1.0 to 5.0 mM. Therefore, this approach allowed the development of a simple, point of care in the medical field, disposable electrochemical uric acid biosensor.

• YAKHAK HOEJI
• /
• v.23 no.3_4
• /
• pp.173-179
• /
• 1979

A dose, 1g/kg of ethanol produced experimental hyperuricemia in mouse. Ginseng saponins were tested for their ability to alter the hepatic xanthine oxidase activity and the blood level of uric acid in the ethanol-treated mouse. Intraperitoneal injection of ginseng saponin 4mg/kg markedly decreased the xanthine oxidase activity in the ethanol-treated mouse liver. It was also observed that ginseng saponin reduced the blood concentration of uric acid in experimentally induced hyperuricemia by alcohol treatment. In vitro, it was found that a low concentration of ginseng saponin in the reaction mixture incresed the hepatic xanthine oxidase activity, while a high concentration inhibited both enzyme preparations of normal and ethanol treated mice. In contrast with the xanthine oxidase, uricase activity was not influenced by ginseng saponin as well as in vivo. These results suggest there is a possibility that ginseng saponin may have some therapeutic effect on gout and other hyperuricemia syndrome.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.25 no.1
• /
• pp.46-52
• /
• 1996

The present study was undertaken to investigate the effect of the water extract of the puffer fish Fugu xanthopterus(FXH) on the alcohol induced hyperuricemia. The normal group and the FXH treated group showed no sigbificant changes in the levels of blood uric acid but, the blood uric acid significantly decreased in the FXh treated rats with 100mg/kg for two weeks compared to the ethanol treated group. There were no significant changes in the activities of uricase, adenosine deaminase, guanine deaminase, and purine uncleoside phosphorylase, among all the test group. But the activitis of liver xanthine oxidase were recovered to the normal level in ethanol +FXH treated group comparing to the ethanol treated group. Furthermore, ethanol+FXH treated rats showed the similar pattern in the levels of blood uric acid and urinary allantoin with normal group. These results indicate that the decreased blood uric acid by the FXH treatment of the alcohol induced hyperuricemia rats may result from decreased activity of hepatic xanthine oxidase.

• YAKHAK HOEJI
• /
• v.30 no.2
• /
• pp.62-67
• /
• 1986

It was attempted to observe the effect of garlic on the hepatic purine metabolizing enzymes in this study. The activities of adenosine deaminase, guanase, and uricase in rats were not changed significantly following the feeding of 5% garlic juice. Whereas, garlic juice inhibited significantly the hepatic xanthine oxidase activity compared to control group with the lapse of treated-period. The orate level of serum in rats was significantly decreased by the treatment of garlic juice. The above inhibitory effect of garlic was greater in boiled garlic juice than fresh garlic juice-treated group. These results indicated that, according to the chemical properties of allicin which is unstable in heat, other components than allicin in garlic may regulate the hepatic purine metabolizing enzymes.

• Journal of Environmental Health Sciences
• /
• v.19 no.2
• /
• pp.69-77
• /
• 1993

In the present study, the comparison of liver damage in carbon tetrachloride (CCl$_4$)-treated rats with that those pretreated with ethanol and an effect of liver injury on the serum and liver xanthine oxidase (XOD) activity were evaluated. The increasing rate of liver weight per body wt., the levels of serum alanine aminotransferase, and the decreasing rate of hepatic glucose-6-phosphatase activity and the protein contents in the liver cell were higher in carbon tetrachloride-treated animals pretreated with ethanol than the carbon tetrachloride-treated group. Especially, the histopathological findings also showed more severe liver damage in the ethanol-pretreated rats than the rats treated with carbon tetrachloride only. In such a experimental condition the xanthine oxidase activity of serum and liver both of carbon tetrachloride-treated rats and those pretreated with ethanol were higher than that of each control group. And the increasing rate of xanthine oxidase enzyme activity to the control group was higher in carbon tetrachloride-treated group pretreated with ethanol than those treated with CCl$_4$. In addition, the heptic uricase activity and the serum levels of uric acid were more increased in carbon tetrachloride-treated group pretreated with ethanol than those in the CCl$_4$-treated rats. On the other hand, there were no statistical differences in hepatic catalase and glutathione S-transferase activities between the CCl$_4$-treated rats and those pretreated with ethanol. In conclusion, it is assumed that the more severe liver damage in ethanol pretreated rats would be due to oxygen free radical produced by the xanthine oxidase system.