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 on to 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 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, 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.

Development and characterization of mitochondrial membrane affinity chromatography columns derived from skeletal muscle and platelets for the study of mitochondrial transmembrane proteins

“…The mitochondria was isolated as previously described [18] using a Mitochondria Isolation Kit for Cultured Cells from Abcam and Miltenyi Biotec.…”

“…Mitochondria obtained from monkey muscle tissue or from human platelets were solubilized using a previously described protocol [18,19]. Briefly, the mitochondria pellets were suspended in 10 ml of Tris buffer [10 mM, pH 7.4] containing 2% (w/v) CHAPS, 10% glycerol, 500 mM NaCl, 5 mM 2-mercaptoethanol, 100 μM benzamidine, 1:100 dilution of protease inhibitor cocktail, 50 μg/ml TPCK, 100 μM PMSF and 100 μM ATP for 18 h at 4 °C.…”

“…The functionality of these columns was validated by characterization of TSPO involving determination of the binding affinity of dipyridamole (DIPY) and establishing the screening for TSPO ligands as previously reported [18]. In addition, F-ATPase/ATP synthase activity was demonstrated on the MMAC-Muscle column.…”

“…Previously, we had demonstrated that mitochondrial membrane fragments from cultured cells can be immobilized onto a stationary phase resulting in a functional mitochondrial membrane affinity chromatography (MMAC) column [18]. The co-immobilization of both OMM and IMM in MMAC column was confirmed by characterization of translocator protein (TSPO), mitochondrial permeability transition pore and sulfonylurea receptor.…”

Cellular membrane affinity chromatography (CMAC) in drug discovery from complex natural matrices

“…This can be achieved through several experimental ways. Firstly, the immobilization of transmembrane proteins onto IAM particles can be confirmed using confocal microscopy [44]. IAM with immobilized cell membrane fragments is incubated with a ligand known to bind to the target protein.…”