Resolution of the V1 ATPase from Manduca sexta into subcomplexes and visualization of an ATPase-active A3B3EG complex by electron microscopy.
The effect of the ATPase activity of Manduca sexta V(1) ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V(1) were studied. The V(1) was highly activated in the presence of 0.04-0.06% LDAO combined with release of the subunits H, C, and F from the enzyme. Increase of LDAO concentration to 0.1-0.2% caused the characterized subcomplexes A(3)B(3)HEGF and A(3)B(3)EG with a remaining ATPase activity of 52 and 65%, respectively. The hydrolytic-active A(3)B(3)EG subcomplex has been visualized by electron microscopy showing six major masses of density in a pseudo-hexagonal arrangement surrounding a seventh mass. The compositions of the various subcomplexes and fragments of V(1) provide an organization of the subunits in the enzyme in the framework of the known three-dimensional reconstruction of the V(1) ATPase from M. sexta (Radermacher, M., Ruiz, T., Wieczorek, H., and Grüber, G. (2001) J. Struct. Biol. 135, 26-37).
Rizzo
Vincenzo F
VF
Universität des Saarlandes, Fachrichtung 2.5-Biophysik, D-66421 Homburg, Germany.
Coskun
Unal
U
Radermacher
Michael
M
Ruiz
Teresa
T
Armbruster
Andrea
A
Gruber
Gerhard
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003902:Detergents / Q000737:chemistry; D004123:Dimethylamines / Q000737:chemistry; D046911:Macromolecular Substances; D018402:Manduca / Q000201:enzymology*; D008854:Microscopy, Electron; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D003902:Detergents; D004123:Dimethylamines; D046911:Macromolecular Substances; D021122:Protein Subunits; C014518:dodecyldimethylamine oxide; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases