Three-dimensional structure and subunit topology of the V-1 ATPase from Manduca sexta midgut

Autor(en): Gruber, G
Radermacher, M
Ruiz, T
Godovac-Zimmermann, J
Canas, B
Kleine-Kohlbrecher, D
Huss, M 
Harvey, WR
Wieczorek, H 
Stichwörter: 3-DIMENSIONAL RECONSTRUCTION; Biochemistry & Molecular Biology; CRYOELECTRON MICROSCOPY; ELECTRON-MICROSCOPY; ESCHERICHIA-COLI; F1 ADENOSINE-TRIPHOSPHATASE; GAMMA-SUBUNIT; MOLECULAR ARCHITECTURE; TOBACCO HORNWORM MIDGUT; V-TYPE ATPASE; VACUOLAR-TYPE ATPASE
Erscheinungsdatum: 2000
Herausgeber: AMER CHEMICAL SOC
Journal: BIOCHEMISTRY
Volumen: 39
Ausgabe: 29
Startseite: 8609
Seitenende: 8616
Zusammenfassung: 
The three-dimensional structure of the Manduca sexta midgut V-1 ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V-1 complex has a barrel-like structure Il nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V-1 complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl2-induced disulfide formation. Treatment of V-1 with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V-1 decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl2 was added to V-1 in the presence of CaADP, the cross-linked products A-E-F and B-H were generated. In experiments where CuCl2 was added after preincubation of CaATP, the cross-linked products E-F and E-G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.
ISSN: 00062960
DOI: 10.1021/bi000103u

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