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11.
Purification and Properties of Component B of 2,4,5-Trichlorophenoxyacetate Oxygenase from Pseudomonas cepacia AC1100 总被引:3,自引:1,他引:2 下载免费PDF全文
Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2). 相似文献
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Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp. 总被引:5,自引:0,他引:5 下载免费PDF全文
Tetrachloro-p-hydroquinone (TeCH) is the first intermediate in pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723. We previously purified a PCP hydroxylase that oxidized PCP to TeCH. Subsequently, we identified the reductive dehalogenation of TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone in a cell extract with the reduced form of glutathione as the reducing agent under anaerobic conditions. Here we report the purification of a TeCH reductive dehalogenase that reductively dehalogenated TeCH to trichlorohydroquinone and then to dichlorohydroquinone. The enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, and phenyl-agarose, anion-exchange, and gel filtration column chromatographies. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the protein has a molecular weight of about 30,000; nondenaturing polyacrylamide gel electrophoresis analysis suggests that the native enzyme exists as a dimer. The enzyme used glutathione but not NADPH, NADH, dithiothreitol, or ascorbic acid as the reducing agent. The optimal pH was close to neutral. 相似文献
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C Q Xun C M Ensor H H Tai 《Biochemical and biophysical research communications》1991,177(3):1258-1265
Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and [35S]methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism. 相似文献
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Biodegradation of triiodophenol by cell-free extracts of a pentachlorophenol-degrading Flavobacterium sp 总被引:5,自引:0,他引:5
Pentachlorophenol (PCP) degrading Flavobacterium sp. ATCC 39723 was found to degrade other polyhalogenated phenolic compounds, including triiodophenol, tribromophenol, and trichlorophenol. Each compound was able to induce the degradation of the other compounds. A PCP Flavobacterium sp. mutant, F-2, was unable to degrade any of the halogenated compounds. The results suggest that all of the polyhalogenated phenols were degraded by the same enzyme system. This observation led us to exploit the sensitive leuco crystal violet assay, which measures the iodide released from triiodophenol. Cell free extracts from PCP-induced cells were able to release iodide from triiodophenol. The reaction required NADPH and oxygen. 相似文献
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Xinhuai Dong Xuan Huang Zhicheng Yao Yun Wu Delin Chen Chahui Tan Jiajie Lin Danrui Zhang Yiwen Hu Jueheng Wu Guohong Wei Xun Zhu 《Journal of cellular and molecular medicine》2020,24(14):7884-7895
Tumour‐associated macrophages (TAMs), which possess M2‐like characters and are derived from immature monocytes in the circulatory system, represent a predominant population of inflammatory cells in solid tumours. TAM infiltration in tumour microenvironment can be used as an important prognostic marker in many cancer types and is a potential target for cancer prevention or treatment. VEGI‐251 not only is involved in the inhibition of tumour angiogenesis, but also participates in the regulation of host immunity. This work aimed to investigate the involvement of VEGI‐251 in the regulation of specific antitumour immunity. We found that recombinant human VEGI‐251(rhVEGI‐251) efficiently mediated the elimination of TAMs in tumour tissue in mice, and induced apoptosis of purified TAMs in vitro. During this process, caspase‐8 and caspase‐3 were activated, leading to PARP cleavage and apoptosis. Most importantly, we further elucidated the mechanism underlying VEGI‐251‐triggered TAM apoptosis, which suggests that ASK1, an intermediate component of the VEGI‐251, activates the JNK pathway via TRAF2 in a potentially DR3‐dependent manner in the process of TAM apoptosis. Collectively, our findings provide new insights into the basic mechanisms underlying the actions of VEGI‐251 that might lead to future development of antitumour therapeutic strategies using VEGI‐251 to target TAMs. 相似文献
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An exploration of the role of Sertoli cells on fetal testis development using cell ablation strategy
Yu‐Qian Wang Jin‐Mei Cheng Qing Wen Ji‐Xin Tang Jian Li Su‐Ren Chen Yi‐Xun Liu 《Molecular reproduction and development》2020,87(2):223-230
Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh‐Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α‐smooth muscle actin‐labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3β‐HSD double‐positive fetal LCs could be observed in Amh‐Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3β‐HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs’ central role in fetal testis development. 相似文献