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

Abstract

Mitochondrial membrane fragments from human platelets and monkey skeletal muscles were successfully immobilized onto immobilized artificial membrane chromatographic support for the first time, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. These columns were validated by characterization of translocator protein (TSPO), where multiple concentrations of dipyridamole were run and the binding affinities (Kd) determined. Further, the relative ranking data of TSPO ligands was consistent with previously reported rankings for both, the platelet (MMAC-Platelet) and the skeletal muscle (MMAC-Muscle) column (dipyridamole>PK11195>protoporphyrin IX>rotenone). The functional immobilization of the F-ATPase/ATP synthase was demonstrated on MMAC-Muscle column. Online hydrolysis of ATP to ADP and synthesis of ATP from ADP were both demonstrated on the MMAC-Muscle column. Hydrolysis of ATP to ADP was inhibited by oligomycin A with an IC50 of 40.2±13.5nM (∼60% reduction in ATP hydrolysis, p<0.001), similar to previously reported values. Additionally, the Michaelis-Menten constant (Km) for ADP was found to be 1525±461μM based on the on column dose-dependent increase in ATP production.