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首页> 外文期刊>Biochemistry >ATP-dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity
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ATP-dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity

机译：ATP依赖性人类红细胞谷胱甘肽-结合物转运蛋白。 I.纯化，光亲和标记和ATPase活性的动力学特征

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Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (K-d = 2 mu M), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching: of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well. [References: 24]
机译：二硝基苯基S-谷胱甘肽（DNP-SG）ATPase是在红细胞和其他组织中表达的38 kDa膜蛋白。尽管已在几种结构上不相关的两亲离子的存在下证明了DNP-SG ATPase催化的ATP水解刺激作用，但该蛋白的结构和功能特性尚未明确。在本研究中，我们已经开发了一种用于纯化DNP-SG ATPase的改进方案，并研究了其动力学和底物结合特性。纯化步骤基于在ATP存在下从DNP-SG亲和树脂中高度特异性地洗脱38 kDa蛋白的方法。不能使用ADP或腺苷5'-β-γ-亚甲基三磷酸酯（亚甲基-ATP）（ATP的不可水解类似物）洗脱蛋白质。已发现弱碱性蒽环类化学疗法药物阿霉素（DOX）是刺激该酶水解ATP的首选活化剂。使用8-叠氮基-ATP光亲和标记和三硝基苯基（TNP）-ATP（ATP的荧光类似物）的结合证明了ATP与酶的结合。 DNP-SG ATPase（38 kDa）的光亲和性标记相对于8-叠氮基ATP（K-d = 2μM）是饱和的，表明该酶能够与ATP特异性结合。 DNP-SG结合从纯化过程本身就很明显，并且通过猝灭色氨酸荧光也可以证明。色氨酸荧光淬灭的结果以及放射性同位素结合研究表明，DOX也与纯化的蛋白结合。 [参考：24]

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