ATP-Dependent Human Erythrocyte Glutathione-Conjugate Transporter. II. Functional Reconstitution of Transport Activity
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- 作者：Sanjay Awasthi ; Sharad S. Singhal ; Slawomir Pikula ; John T. Piper ; Sanjay K. Srivastava ; Robert T. Torman ; Joanna Bandorowicz-Pikula ; James T. Lin ; Shivendra V. Singh ; Piotr Zimniak ; and Yogesh C. Awa
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：April 14, 1998
- 年：1998
- 卷：37
- 期：15
- 页码：5239 - 5248
- 全文大小：138K
- 年卷期：v.37,no.15(April 14, 1998)
- ISSN：1520-4995
文摘
Purified dinitrophenyl S-glutathione(DNP-SG) ATPase was reconstituted into artificialliposomes prepared from soybean asolectin. Electron micrographyconfirmed the formation of unilamellarvesicles with an average radius of 0.25 
m. Intravesicularvolume estimated by incorporation of radiolabledinulin into the vesicles was found to be 19.7 ± 1.3 L/mLreconstitution solution. Accumulation of theglutathione-conjugate of CDNB, DNP-SG, and of doxorubicin (DOX) in theproteoliposomes was increasedin the presence of ATP as compared to equimolar ADP or adenosine5'-[
,
-methylene]triphosphatetetralithium. ATP-dependent transmembrane movement of DOX andDNP-SG into DNP-SG ATPase-reconstituted vesicles was saturable with respect to time, sensitive tothe osmolarity of the assay medium,and temperature dependent. The energy of activation was found tobe 12 and 15 kcal/mol for DNP-SGand DOX, respectively. Optimal temperature for transport was 37
C. Saturable transport was demonstratedfor DNP-SG (Vmax of 433 ± 20 nmol/min/mg ofprotein, KmATP = 2.4 ± 0.3 mM andKmDNP-SG = 36 ±5 M) as well as DOX (Vmax = 194 ± 19nmol/min/mg of protein, KmATP = 2.5 ± 0.6 mMand KmDOX= 2.4 ± 0.7 M). The kinetic data for both DNP-SG and DOXtransport were consistent with a randombi-bi sequential reaction mechanism. DOX was found to be acompetitive inhibitor of DNP-SG transportwith Kis of 1.2 ± 0.2 M and DNP-SG wasfound to be a competitive inhibitor of DOX transport withKis of 13.3 ± 2.6 M.
m. Intravesicularvolume estimated by incorporation of radiolabledinulin into the vesicles was found to be 19.7 ± 1.3 L/mLreconstitution solution. Accumulation of theglutathione-conjugate of CDNB, DNP-SG, and of doxorubicin (DOX) in theproteoliposomes was increasedin the presence of ATP as compared to equimolar ADP or adenosine5'-[,-methylene]triphosphatetetralithium. ATP-dependent transmembrane movement of DOX andDNP-SG into DNP-SG ATPase-reconstituted vesicles was saturable with respect to time, sensitive tothe osmolarity of the assay medium,and temperature dependent. The energy of activation was found tobe 12 and 15 kcal/mol for DNP-SGand DOX, respectively. Optimal temperature for transport was 37C. Saturable transport was demonstratedfor DNP-SG (Vmax of 433 ± 20 nmol/min/mg ofprotein, KmATP = 2.4 ± 0.3 mM andKmDNP-SG = 36 ±5 M) as well as DOX (Vmax = 194 ± 19nmol/min/mg of protein, KmATP = 2.5 ± 0.6 mMand KmDOX= 2.4 ± 0.7 M). The kinetic data for both DNP-SG and DOXtransport were consistent with a randombi-bi sequential reaction mechanism. DOX was found to be acompetitive inhibitor of DNP-SG transportwith Kis of 1.2 ± 0.2 M and DNP-SG wasfound to be a competitive inhibitor of DOX transport withKis of 13.3 ± 2.6 M.