title: The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.
abstract: 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.
authors.affiliation: Department of Natural Sciences, Institute of Mathematics and Natural Sciences, Tallinn University, 10120 Tallinn, Estonia; Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: K-L
authors.initials: KL
authors.lastname: Habicht
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: N S
authors.initials: NS
authors.lastname: Singh
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: F E
authors.initials: FE
authors.lastname: Indig
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: I W
authors.initials: IW
authors.lastname: Wainer
authors.affiliation: Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: R
authors.initials: R
authors.lastname: Moaddel
authors.affiliation: Department of Natural Sciences, Institute of Mathematics and Natural Sciences, Tallinn University, 10120 Tallinn, Estonia. Electronic address: ruth.shimmo@tlu.ee.
authors.forename: R
authors.initials: R
authors.lastname: Shimmo
mesh_terms: D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008567:Membranes, Artificial; D018613:Microscopy, Confocal; D051336:Mitochondrial Membranes / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008024:Ligands; D008565:Membrane Proteins; D008567:Membranes, Artificial
keywords: Mitochondrial membrane affinity chromatography; Translocator protein