The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.
Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.
['Habicht|K-L|KL|', 'Singh|N S|NS|', 'Indig|F E|FE|', 'Wainer|I W|IW|', 'Moaddel|R|R|', 'Shimmo|R|R|']
D001665:Binding Sites D002846:Chromatography, Affinity D057809:HEK293 Cells D006801:Humans D008024:Ligands D008565:Membrane Proteins D008567:Membranes, Artificial D018613:Microscopy, Confocal D051336:Mitochondrial Membranes
Mitochondrial membrane affinity chromatography Translocator protein