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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geprüft am 17.05.2024