Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl beta-D-maltoside.
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 micromol/min/mg, 2.5-fold higher than that previously reported. ATPase activity was 80% inhibited by the F0-reactive reagent dicyclohexylcarbodiimide, indicating that F1-F0 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 approximately 30-min lag phase.
Hazard
Andrea
A
Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA.
Montemagno
Carlo
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D001190:Arylsulfonates / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism*
D001190:Arylsulfonates; D003902:Detergents; D005456:Fluorescent Dyes; D005960:Glucosides; D008081:Liposomes; D001436:Bacteriorhodopsins; C040358:dodecyl maltoside; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; C005047:pyranine