Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl β-D-maltoside

被引:33
作者
Hazard, A
Montemagno, C
机构
[1] Univ Calif Los Angeles, Dept Biomed Engn, Los Angeles, CA 90095 USA
[2] Cornell Univ, Dept Biol & Environm Engn, Ithaca, NY 14853 USA
关键词
ATP synthesis; oxidative phosphorylation; F1F0 ATP synthase; pyranine; bacteriorhodopsin; reconstitution; proteoliposomes; proton pumping;
D O I
10.1016/S0003-9861(02)00469-1
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
Here we report a fast, simple purification for thermophilic F1F0 ATP synthase (TF1F0) that utilizes a cocktail of stabilizing reagents and the detergent n-dodecyl beta-D-maltoside to yield enzyme with an ATPase activity of 41 mumol/min/mg, 2.5-fold higher than that previously reported. ATPase activity was 80% inhibited by the F-0-reactive reagent dicyclohexylcarbodiimide, indicating that F-1-F-0 interactions were largely intact. To measure ATP-driven proton pumping activity, purified TF1F0 was incorporated into liposomes, and the ATP-induced change in internal pH was measured using the fluorescent probe pyranine. In the presence of valinomycin, a maximum ATP-driven DeltapH of 0.8 units was obtained. To measure ATP synthesis activity, TF1F0 was incorporated into liposomes with the light-dependent proton pump bacteriorhodopsin. Proteoliposomes were illuminated to generate an electrochemical gradient, after which ADP and inorganic phosphate were added to initiate ATP synthesis. A steady state ATP synthesis activity of 490 nmol/min/mg was achieved after an initial similar to30-min lag phase. (C) 2002 Elsevier Science (USA). All rights reserved.
引用
收藏
页码:117 / 124
页数:8
相关论文
共 35 条
[1]   LINEAR RELATIONS BETWEEN PROTON CURRENT AND PH GRADIENT IN BACTERIORHODOPSIN LIPOSOMES [J].
ARENTS, JC ;
VANDEKKEN, H ;
HELLINGWERF, KJ ;
WESTERHOFF, HV .
BIOCHEMISTRY, 1981, 20 (18) :5114-5123
[2]   Mechanism of the F1F0-type ATP synthase, a biological rotary motor [J].
Capaldi, RA ;
Aggeler, R .
TRENDS IN BIOCHEMICAL SCIENCES, 2002, 27 (03) :154-160
[3]  
DENCHER NA, 1982, METHOD ENZYMOL, V88, P5
[4]  
DUNCAN TM, 1985, J BIOL CHEM, V260, P4901
[5]  
FILLINGAME RH, 1986, METHOD ENZYMOL, V126, P545
[6]   ATP synthase from bovine heart mitochondria: Reconstitution into unilamellar phospholipid vesicles of the pure enzyme in a functional state [J].
Groth, G ;
Walker, JE .
BIOCHEMICAL JOURNAL, 1996, 318 :351-357
[7]   CHARACTERIZATION OF THE BINDING OF VALINOMYCIN TO BACTERIORHODOPSIN [J].
HASSELBACHER, CA ;
DEWEY, TG .
PHOTOSYNTHESIS RESEARCH, 1986, 8 (01) :79-86
[8]  
HAZARD AL, 1994, J BIOL CHEM, V269, P427
[9]   BACTERIORHODOPSIN IN LIPOSOMES .2. EXPERIMENTAL-EVIDENCE IN SUPPORT OF A THEORETICAL-MODEL [J].
HELLINGWERF, KJ ;
ARENTS, JC ;
SCHOLTE, BJ ;
WESTERHOFF, HV .
BIOCHIMICA ET BIOPHYSICA ACTA, 1979, 547 (03) :561-582
[10]  
HJELMELAND LM, 1990, METHOD ENZYMOL, V182, P253