DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES
红细胞中异生物质的解毒
基本信息
- 批准号:2391935
- 负责人:
- 金额:$ 18.91万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1984
- 资助国家:美国
- 起止时间:1984-07-01 至 1999-03-31
- 项目状态:已结题
- 来源:
- 关键词:P glycoprotein adenosinetriphosphatase affinity chromatography biological transport chemical conjugate detoxification doxorubicin enzyme activity enzyme mechanism erythrocyte membrane glutathione human subject immunoaffinity chromatography laboratory rabbit lipid peroxides liposomes membrane transport proteins molecular cloning nucleic acid probes protein purification protein sequence protein structure function
项目摘要
During the funded year of this project, we have characterized a
transporter involved in ATP-dependent primary active transport of
glutathione (GSH)-conjugates in human erythrocyte membrane and
designated it as dinitrophenyl S-glutathione (Dnp-SG) ATPase
because the use of Dnp-SG as a model substrate. Subsequently we
showed that Dnp-SG ATPase was ubiquitous in human cell plasma
membranes and that in addition to GSH-conjugates it was also
involved in the ATP-dependent transport of bilirubin-conjugates,
leukotrienes, and structurally unrelated compounds such as
doxorubicin and other substrates of P-glycoprotein, a well
characterized ATP-dependent pump overexpresed in multidrug
resistance cancer cells. These studies, for the first time
demonstrated the presence of an extremely versatile transporter
(distinct from P-glycoprotein) in human cells which could actively
transport diverse group of xenobiotics, drugs, and their phase I
and phase II metabolites. To establish a unifying theme for the
mechanisms of transport of such structually diverse compounds by
Dnp-SG ATPase, studies are proposed in this application for its
structural and functional characterization. Dnp-SG ATPase will be
purified from human erytocytes and other tissues by Dnp-SG affinity
chromatography and immunoaffinity chromatography to determine its
structural and functional properties. The amino acid sequences of
the peptide fragments of Dnp-SG ATPase generated by CNBr cleavage
and isolated by HPLC and/or in SDS gels followed by transblotting
on P-PVDF membranes will be determined. These sequences will be
used to design and synthesize nucleotide probes to clone and
sequence the cDNA of Dnp-SG ATPase to deduce its primary structure.
Recombinant Dnp-SG ATPase will be prepared by expressing it in E.
coli and/or other suitable vectors to get sufficient protein for
its structural and functional characterization. Antibodies against
Dnp-SG ATPase will be usssl in the alternate approaches for
cloning. Possible genomic heterdgereity at Dnp-SG ATPase locus will
be investigated to examine the existence of other related
transporters at this locus. The kinetics of the ATP hydrolyzing
activity of Dnp-SG ATPase stimulated by GSH-conjugates of
xenobiotics and toxic products of lipid peroxidation such as 4-
hydroxynonenal (4-HNE), and the substrates of P-glycoprotein (e.g.
doxorubicin, vincristine) will be studied. Also the kinetics and
mechanisms of the ATP-dependent transport of these compounds in the
inside out vesicles (IOVs) prepared from erythrocyte membranes and
in reconstituted proteoliposomes with native recombinant Dnp-SG
ATPase will be studied. We will test the hypothesis whether Dnp-SG
ATPase is a mediator of doxorubicin transport and hence resistance
of P glycoprotein negative, doxorubicin resistant small cell lung
cancer cell lines developed by us from parental NCI H-69 cell line.
Studies proposed in this project will define the role of Dnp-SG
ATPase in the protection mechanisms against structurally diverse
xenobiotics and toxic endobiotics (such as 4-HNE), and will test
the hypothesis that Dnp-SG ATPase may be involved in the mechanisms
of drug resistance of cancer cells, particularly those which do not
express P glycoprotein.
在这项计划的资助年度内,我们的特点是
转运体参与ATP依赖的初级主动转运
人红细胞膜谷胱甘肽(GSH)结合物
命名为二硝基苯基S-谷胱甘肽ATPase
因为使用了DNP-SG作为模型衬底。随后,我们
结果表明,DNP-SG ATPase在人细胞质中普遍存在
膜,除了GSH偶联物外,它还
参与依赖于ATP的胆红素结合物的运输,
白三烯和结构上无关的化合物,如
阿霉素等P-糖蛋白底物,一井
特征性的ATP依赖泵在多药中过度表达
耐药癌细胞。这些研究是第一次
展示了一种非常多功能的运输机的存在
(有别于P-糖蛋白)在人类细胞中可以活跃地
运输不同种类的外源物质、药物及其I期
和第二相代谢物。建立一个统一的主题,
这种结构不同的化合物的运输机制
DNP-SG ATPase,建议在这一应用中对其进行研究
结构和功能表征。DNP-SG ATPase将是
DNP-SG亲和纯化人红细胞及其他组织
层析法和免疫亲和层析法测定其
结构和功能特性。其氨基酸序列
CNBr裂解产生的DNP-SG-ATPase的多肽片段
用高效液相色谱法和/或在转印后的十二烷基硫酸钠凝胶中分离
将对P-PVDF膜进行测定。这些序列将是
用于设计和合成核苷酸探针以克隆和
对DNP-SG ATPase基因进行测序,推测其一级结构。
重组DNP-SG-ATPase在E.
Coli和/或其他合适的载体以获得足够的蛋白质
它的结构和功能表征。抗病毒抗体
DNP-SG ATPase将用于以下替代方法
克隆。DNP-SG ATPase基因座可能存在的基因组杂合性将
被调查以检查是否存在其他相关的
这个地点的传送者。三磷酸腺苷水解酶的动力学
谷胱甘肽结合物对DNP-SG-ATPase活性的影响
异生物质和脂质过氧化的有毒产物,如4-
羟基壬烯醛(4-HNE)和P-糖蛋白底物(例如
阿霉素、长春新碱)将被研究。此外,动力学和
这些化合物在体内依赖于ATP转运的机制
由红细胞膜和红细胞膜制备的内翻式囊泡
在含有天然重组DNP-SG的重组蛋白脂质体中
将对ATPase进行研究。我们将检验假设DNP-SG是否
ATPase是阿霉素转运的中介物,因此产生耐药性
P糖蛋白阴性、阿霉素耐药小细胞肺
我们从亲代NCI H-69细胞系中培育出癌细胞系。
本项目中提出的研究将确定DNP-SG的作用
ATPase在结构多样性保护机制中的作用
外源生物和有毒内生生物(如4-HNE),并将测试
DNP-SG-ATPase可能参与发病机制的假说
癌细胞的抗药性,特别是那些不耐药的癌细胞
表达P糖蛋白。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
YOGESH Chandra AWASTHI其他文献
YOGESH Chandra AWASTHI的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('YOGESH Chandra AWASTHI', 18)}}的其他基金
Protection of Oxidant Toxicity by Glutathione S Transferases
谷胱甘肽 S 转移酶对氧化剂毒性的保护
- 批准号:
7173030 - 财政年份:2003
- 资助金额:
$ 18.91万 - 项目类别:
Protection of Oxidant Toxicity by Glutathione S Transferases
谷胱甘肽 S 转移酶对氧化剂毒性的保护
- 批准号:
7337306 - 财政年份:2003
- 资助金额:
$ 18.91万 - 项目类别:
相似海外基金
MOLECULAR CHARACTERIZATION OF THE SODIUM POTASSIUM TRANSPORT ADENOSINETRIPHOSPHATASE
钠钾转运腺苷三磷酸酶的分子表征
- 批准号:
7461764 - 财政年份:1974
- 资助金额:
$ 18.91万 - 项目类别:
MOLECULAR CHARTERIZATION OF THE SODIUM-POTASSIUM TRANSPORT ADENOSINETRIPHOSPHATASE
钠钾转运腺苷三磷酸酶的分子表征
- 批准号:
7352845 - 财政年份:1973
- 资助金额:
$ 18.91万 - 项目类别:
Molecular Characterization of the Sodium-Potassiumtransport Adenosinetriphosphatase
钠钾转运三磷酸腺苷酶的分子表征
- 批准号:
7301506 - 财政年份:1973
- 资助金额:
$ 18.91万 - 项目类别:
Continuing Grant
MOLECULAR CHARACTERIZATION OF THE SODIUM-POTASSIUM TRANSPORT ADENOSINETRIPHOSPHATASE
钠钾转运腺苷三磷酸酶的分子表征
- 批准号:
7243716 - 财政年份:1972
- 资助金额:
$ 18.91万 - 项目类别:
MOLECULAR CHARACTERIZATION OF THE SODIUM-POTASSIUM TRANSPORT ADENOSINETRIPHOSPHATASE
钠钾转运腺苷三磷酸酶的分子表征
- 批准号:
7138222 - 财政年份:1971
- 资助金额:
$ 18.91万 - 项目类别:
Molecular Characterization of the Sodium-Potassium Transport Adenosinetriphosphatase
钠钾转运三磷酸腺苷酶的分子表征
- 批准号:
6928993 - 财政年份:1969
- 资助金额:
$ 18.91万 - 项目类别:
Adenosinetriphosphatase Genesis in Bone Marrow Cells
骨髓细胞中腺苷三磷酸酶的发生
- 批准号:
64B2295 - 财政年份:1964
- 资助金额:
$ 18.91万 - 项目类别:
Adenosinetriphosphatase and sugar Transport Mechanism
三磷酸腺苷酶和糖转运机制
- 批准号:
6216854 - 财政年份:1962
- 资助金额:
$ 18.91万 - 项目类别:
Bone Marrow Cells and Relation to Adenosinetriphosphatase Activity
骨髓细胞及其与三磷酸腺苷酶活性的关系
- 批准号:
6216803 - 财政年份:1962
- 资助金额:
$ 18.91万 - 项目类别:














{{item.name}}会员




