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J. Biol. Chem., Vol. 277, Issue 37, 33906-33912, September 13, 2002

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A Functionally Active Human F₁F₀ ATPase Can be Purified by Immunocapture from Heart Tissue and Fibroblast Cell Lines
SUBUNIT STRUCTURE AND ACTIVITY STUDIES^*

Robert Aggeler, Juliana Coons, Steven W. Taylor^§, Soumitra S. Ghosh^§, José J. García^¶, Roderick A. Capaldi, and Michael F. Marusich

From the  Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, ^§ MitoKor, San Diego, California 92121, and the ^¶ Instituto Nacional de Cardiología, Departamento de Bioquímica, Juan Badiano 1 Col. Sección XVI, 14080, México. D. F., México

Human mitochondrial F₁F₀ ATP synthase was isolated with a one-step immunological approach, using a monoclonal antibody against F₁ in a 96-well microplate activity assay system, to establish a method for fast high throughput screening of inhibitors, toxins, and drugs with very small amounts of enzyme. For preparative purification, mitochondria from human heart tissue as well as cultured fibroblasts were solubilized with dodecyl--D-maltoside, and the F₁F₀ was isolated with anti-F₁ monoclonal antibody coupled to protein G-agarose beads. The immunoprecipitated F₁F₀ contained a full complement of subunits that were identified with specific antibodies against five of the subunits (, , OSCP, d, and IF₁) and by MALDI-TOF and/or LC/MS/MS for all subunits except subunit c, which could not be resolved by these methods because of the limits of detection. Microscale immunocapture of F₁F₀ from detergent-solubilized mitochondria or whole cell fibroblast extracts was performed using anti-F₁ monoclonal antibody immobilized on 96-well microplates. The captured complex V displayed ATP hydrolysis activity that was fully oligomycin and inhibitor protein IF₁-sensitive. Moreover, IF₁ could be co-isolated with F₁F₀ when the immunocapture procedure was carried out at pH 6.5 but was absent when the ATP synthase was isolated at pH 8.0. Immunocaptured F₁F₀ lacking IF₁ could be inhibited by more than 90% by addition of recombinant inhibitor protein, and conversely, F₁F₀ containing IF₁ could be activated more than 10-fold by brief exposure to pH 8.0, inducing the release of inhibitor protein. With this microplate system an ATP hydrolysis assay of complex V could be carried out with as little as 10 ng of heart mitochondria/well and as few as 3 × 10⁴ cells/well from fibroblast cultures. The system is therefore suitable to screen patient-derived samples for alterations in amount or functionality of both the F₁F₀ ATPase and IF₁.

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