Efficient purification and reconstitution of P-glycoprotein for functional and structural studies

被引:59
作者
Dong, MQ [1 ]
Penin, F [1 ]
Baggetto, LG [1 ]
机构
[1] INST BIOL & CHIM PROT, CNRS, UPR 412, F-69367 LYON 07, FRANCE
关键词
D O I
10.1074/jbc.271.46.28875
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Plasma membrane P-glycoprotein is known as an ATP-dependent drug efflux pump that confers multidrug resistance to tumor cells. None of the reported purification procedures worked properly for our P-glycoprotein overproducing cell lines, i.e. murine lymphoid leukemia P388/ADR25, rat hepatoma AS30-D/COL10 and human lymphoblastic leukemia CEM/VLB5 cells. We have thus developed a general procedure for efficient purification of P-glycoprotein by combining solubilization with sodium dodecyl sulfate and chromatography on ceramic hydroxyapatite. This procedure was successful for the three cell lines and yielded 70% of the P-glycoprotein present in the starting plasma membranes with more than 99% purity. After exchanging sodium dodecyl sulfate into dodecyl maltoside and reconstitution into liposomes, purified P-glycoprotein exhibited a specific ATPase activity of about 200 nmol/min/mg, which was very similar to that obtained for P-glycoprotein solubilized and purified with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid. This ATPase activity was sensitive to orthovanadate inhibition and stimulated by verapamil and other drugs. More importantly, drug transport properties of the reconstituted P-glycoprotein were comparable with those of P-glycoprotein embedded in plasma membranes. Since it is virtually devoid of lipids, this preparation is suitable for both functional and structural investigations.
引用
收藏
页码:28875 / 28883
页数:9
相关论文
共 50 条
[1]   PURIFICATION AND RECONSTITUTION OF FUNCTIONAL HUMAN P-GLYCOPROTEIN [J].
AMBUDKAR, SV .
JOURNAL OF BIOENERGETICS AND BIOMEMBRANES, 1995, 27 (01) :23-29
[2]   PARTIAL-PURIFICATION AND RECONSTITUTION OF THE HUMAN MULTIDRUG-RESISTANCE PUMP - CHARACTERIZATION OF THE DRUG-STIMULATABLE ATP HYDROLYSIS [J].
AMBUDKAR, SV ;
LELONG, IH ;
ZHANG, JP ;
CARDARELLI, CO ;
GOTTESMAN, MM ;
PASTAN, I .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (18) :8472-8476
[3]   RECONSTITUTION OF ATP-DEPENDENT AMINOPHOSPHOLIPID TRANSLOCATION IN PROTEOLIPOSOMES [J].
AULAND, ME ;
ROUFOGALIS, BD ;
DEVAUX, PF ;
ZACHOWSKI, A .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (23) :10938-10942
[4]  
BAGGETTO LG, 1987, J BIOL CHEM, V262, P9535
[5]  
BAUBICHONCORTAY H, 1994, J BIOL CHEM, V269, P22983
[6]   PURIFICATION AND FUNCTIONAL RECONSTITUTION OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) [J].
BEAR, CE ;
LI, CH ;
KARTNER, N ;
BRIDGES, RJ ;
JENSEN, TJ ;
RAMJEESINGH, M ;
RIORDAN, JR .
CELL, 1992, 68 (04) :809-818
[7]  
BLOOM H, 1987, ELECTROPHORESIS, V8, P93
[8]   DIMERIZATION OF THE P-GLYCOPROTEIN IN MEMBRANES [J].
BOSCOBOINIK, D ;
DEBANNE, MT ;
STAFFORD, AR ;
JUNG, CY ;
GUPTA, RS ;
EPAND, RM .
BIOCHIMICA ET BIOPHYSICA ACTA, 1990, 1027 (03) :225-228
[9]  
BRAIMAN MS, 1987, J BIOL CHEM, V262, P9271
[10]  
DOIGE CA, 1993, BIOCHIM BIOPHYS ACTA, V1146, P65, DOI 10.1016/0005-2736(93)90339-2