Large scale purification of detergent-soluble P-glycoprotein from Pichia pastoris cells and characterization of nucleotide binding properties of wild-type, Walker A, and Walker B mutant proteins

被引:129
作者
Lerner-Marmarosh, N
Gimi, K
Urbatsch, IL
Gros, P
Senior, AE
机构
[1] Univ Rochester, Ctr Med, Dept Biochem & Biophys, Rochester, NY 14642 USA
[2] McGill Univ, Dept Biochem, Montreal, PQ H3G 1Y6, Canada
关键词
D O I
10.1074/jbc.274.49.34711
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
P-glycoprotein (Pgp; mouse MDR3) was expressed in Pichia pastoris, grown in fermenter culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermenter run, Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wildtype mouse MDR3 Pgp were studied using 2'-(3')-O-(2,4,6-trinitrophenyl) adeno sine tri- and diphosphate. Both analogs were found to bind with K-d in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp.
引用
收藏
页码:34711 / 34718
页数:8
相关论文
共 46 条
[11]   The vacuolar H+-Pase of clathrin-coated vesicles is reversibly inhibited by S-nitrosoglutathione [J].
Forgac, M .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (03) :1301-1305
[12]  
GOTTESMAN MM, 1993, CANCER RES, V53, P747
[13]   BIOCHEMISTRY OF MULTIDRUG-RESISTANCE MEDIATED BY THE MULTIDRUG TRANSPORTER [J].
GOTTESMAN, MM ;
PASTAN, I .
ANNUAL REVIEW OF BIOCHEMISTRY, 1993, 62 :385-427
[14]  
GREENBERGER LM, 1993, J BIOL CHEM, V268, P11417
[15]  
Gros P, 1993, Int Rev Cytol, V137C, P169
[16]   Contribution to substrate specificity and transport of nonconserved residues in transmembrane domain 12 of human P-glycoprotein [J].
Hafkemeyer, P ;
Dey, S ;
Ambudkar, SV ;
Hrycyna, CA ;
Pastan, I ;
Gottesman, MM .
BIOCHEMISTRY, 1998, 37 (46) :16400-16409
[17]   ABC TRANSPORTERS - FROM MICROORGANISMS TO MAN [J].
HIGGINS, CF .
ANNUAL REVIEW OF CELL BIOLOGY, 1992, 8 :67-113
[18]   PREPARATION AND PROPERTIES OF 2'(OR 3')-O-(2,4,6-TRINITROPHENYL) ADENOSINE 5'-TRIPHOSPHATE, AN ANALOG OF ADENOSINE-TRIPHOSPHATE [J].
HIRATSUKA, T ;
UCHIDA, K .
BIOCHIMICA ET BIOPHYSICA ACTA, 1973, 320 (03) :635-647
[19]   Structural flexibility of the linker region of human P-glycoprotein permits ATP hydrolysis and drug transport [J].
Hrycyna, CA ;
Airan, LE ;
Germann, UA ;
Ambudkar, SV ;
Pastan, I ;
Gottesman, MM .
BIOCHEMISTRY, 1998, 37 (39) :13660-13673
[20]   Crystal structure of the ATP-binding subunit of an ABC transporter [J].
Hung, LW ;
Wang, IXY ;
Nikaido, K ;
Liu, PQ ;
Ames, GFL ;
Kim, SH .
NATURE, 1998, 396 (6712) :703-707