ADENOSINE TRIPHOSPHATE-DEPENDENT TRANSPORT OF DOXORUBICIN, DAUNOMYCIN, AND VINBLASTINE IN HUMAN TISSUES BY A MECHANISM DISTINCT FROM THE P-GLYCOPROTEIN
ADENOSINE TRIPHOSPHATE-DEPENDENT TRANSPORT OF DOXORUBICIN, DAUNOMYCIN, AND VINBLASTINE IN HUMAN TISSUES BY A MECHANISM DISTINCT FROM THE P-GLYCOPROTEIN
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DOI：
10.1172/jci117102
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发表时间：
1994-03-01
影响因子：
15.9
通讯作者：
AWASTHI, YC
中科院分区：
文献类型：
--
作者：
AWASTHI, S;SINGHAL, SS;AWASTHI, YC
先前的研究表明，一种被称为二硝基苯基-S-谷胱甘肽 ATP 酶 (DNP-SG ATPase) 的人谷胱甘肽缀合物转运蛋白，在除谷胱甘肽缀合物之外的几种两亲性化合物（Singhal、S. S.、R. Sharma、S. Gupta、H. Ahmad、P. Zimniak、A. Radominska、R. Lester 和 Y. C.Awasthi，1991 年。FEBS。《生物化学》杂志，281：255-257。我们现在证明，从人肺和红细胞膜中纯化的 DNP-SG ATP 酶在阿霉素及其代谢物存在的情况下催化 ATP 的水解。由 DNP-SG ATP 酶进行的阿霉素刺激的 ATP 水解相对于阿霉素而言是饱和的（肺酶和红细胞酶的 K-m 分别为 1.2 和 2.8 μM）。抗 DNP-SG ATP 酶的抗体可免疫沉淀阿霉素、其代谢物和谷胱甘肽缀合物刺激的 ATP 水解活性。由红细胞膜制备的内向外囊泡以 ATP 依赖性方式吸收阿霉素、道诺霉素和长春花碱。摄取与时间和囊泡蛋白呈线性关系，依赖于 ATP 和镁，受到重金属盐或加热囊泡的抑制，并且对囊泡的渗透压和方向敏感。该转运的活化能为 13 kcal/mol，对阿霉素和 ATP 均饱和（K-m 值分别为 1.8 μM 和 1.9 mM），并且被谷胱甘肽缀合物以及许多两亲物（例如柔红霉素或长春碱）竞争性抑制。用抗 DNP-SG ATP 酶的抗体包被囊泡后，转运就会减弱。将越来越多的纯化 DNP-SG ATP 酶掺入囊泡中，导致阿霉素转运线性增加。这些研究首次证明，催化阴离子两亲分子（例如谷胱甘肽缀合物）转运的膜蛋白也可以介导弱阳离子抗肿瘤抗生素阿霉素的转运。值得注意的是，转运的 K-m 处于静脉给药阿霉素后人血清中可达到的阿霉素浓度范围内。
Previous studies have demonstrated that a human glutathione conjugate transporter, designated as dinitrophenyl-S-glutathione ATPase(DNP-SG ATPase), catalyzed ATP hydrolysis in the presence of several amphiphilic compounds other than glutathione conjugates (Singhal, S. S., R. Sharma, S. Gupta, H. Ahmad, P. Zimniak, A. Radominska, R. Lester, and Y. C. Awasthi. 1991. FEBS[Fed. Eur. Biochem. Soc] Lett. 281:255-257). We now demonstrate that DNP-SG ATPase purified from human lung and erythrocyte membranes catalyzed the hydrolysis of ATP in the presence of doxorubicin and its metabolites. Doxorubicin-stimulated ATP hydrolysis by DNP-SG ATPase was saturable with respect to doxorubicin (K-m 1.2 and 2.8 mu M for the lung and erythrocyte enzymes, respectively). Antibodies against DNP-SG ATPase immunoprecipitated the ATP hydrolyzing activity stimulated by doxorubicin, its metabolites, and glutathione conjugates. Inside out vesicles prepared from erythrocyte membranes took up doxorubicin, daunomycin, and vinblastine in an ATP-dependent manner. The uptake was linear with respect to time and vesicle protein, was dependent on ATP and magnesium, was inhibited by heavy metal salts or by heating the vesicles, and was sensitive to both osmolarity and orientation of the vesicles. The transport had an activation energy of 13 kcal/mol, was saturable with respect to both doxorubicin and ATP (K-m values of 1.8 mu M and 1.9 mM, respectively), and was competitively inhibited by glutathione conjugates as well as by a number of amphiphiles such as daunomycin or vinblastine. Transport was diminished upon coating the vesicles with antibodies against DNP-SG ATPase. Incorporation of increasing amounts of purified DNP-SG ATPase into the vesicles resulted in a linear increase in transport of doxorubicin. These studies demonstrated for the first time that a membrane protein that catalyzed the transport of anionic amphiphilic molecules such as glutathione conjugates could also mediate the transport of weakly cationic antitumor antibiotic, doxorubicin. Notably, the K-m of transport was in the range of doxorubicin concentration achievable in human serum after intravenous dosing of doxorubicin.