F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase.
A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.
Lutter
R
R
Medical Research Council, Laboratory of Molecular Biology, Cambridge, U.K.
Saraste
M
M
van Walraven
H S
HS
Runswick
M J
MJ
Finel
M
M
Deatherage
J F
JF
Walker
J E
JE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000645:Ammonium Sulfate; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D008929:Mitochondria, Heart / Q000201:enzymology*; D009840:Oligomycins / Q000494:pharmacology*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D003902:Detergents; D005960:Glucosides; D009840:Oligomycins; C040358:dodecyl maltoside; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000645:Ammonium Sulfate