A functionally active human F1F0 ATPase can be purified by immunocapture from heart tissue and fibroblast cell lines. Subunit structure and activity studies.
Human mitochondrial F(1)F(0) ATP synthase was isolated with a one-step immunological approach, using a monoclonal antibody against F(1) 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-beta-d-maltoside, and the F(1)F(0) was isolated with anti-F(1) monoclonal antibody coupled to protein G-agarose beads. The immunoprecipitated F(1)F(0) contained a full complement of subunits that were identified with specific antibodies against five of the subunits (alpha, beta, OSCP, d, and IF(1)) 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 F(1)F(0) from detergent-solubilized mitochondria or whole cell fibroblast extracts was performed using anti-F(1) monoclonal antibody immobilized on 96-well microplates. The captured complex V displayed ATP hydrolysis activity that was fully oligomycin and inhibitor protein IF(1)-sensitive. Moreover, IF(1) could be co-isolated with F(1)F(0) 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 F(1)F(0) lacking IF(1) could be inhibited by more than 90% by addition of recombinant inhibitor protein, and conversely, F(1)F(0) containing IF(1) 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 x 10(4) cells/well from fibroblast cultures. The system is therefore suitable to screen patient-derived samples for alterations in amount or functionality of both the F(1)F(0) ATPase and IF(1).
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA.
Robert
R
Aggeler
Juliana
J
Coons
Steven W
SW
Taylor
Soumitra S
SS
Ghosh
José J
JJ
Garcia
Roderick A
RA
Capaldi
Michael F
MF
Marusich
D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000201:enzymology*; D011233:Precipitin Tests; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D021122:Protein Subunits; D006180:Proton-Translocating ATPases