Determination of free Mg2+ in isolated heart mitochondria using fluorescent probes.
The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored using the fluorescent probe furaptra. The techniques used for loading, calibrating and using furaptra fluorescence to monitor matrix free Mg2+ are described. Furaptra is loaded as the membrane-permeable acetoxymethyl ester which is hydrolyzed to the impermeant acid form by nonspecific esterases. Loading is sufficient in a 20-min period at 25 degrees C to give signals approximately 5-fold greater than nonloaded mitochondria. Uncertainties concerning the apparent dissociation constant value for Mg2+ for entrapped furaptra and the ability of ionophores to equilibrate Mg2+ across the mitochondrial membrane are discussed. We conclude that our best estimate for matrix Mg2+ in isolated mitochondria is 0.5 mM and that it can change significantly with changing Mg2+ ligand availability in the matrix.
Jung
D W
DW
Department of Medical Biochemistry, Ohio State Univesity Medical Center, Columbus.
Brierley
G P
GP
D000818:Animals; D001572:Benzofurans; D002417:Cattle; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D010080:Oxazoles; D013552:Swine
D001572:Benzofurans; D005456:Fluorescent Dyes; D010080:Oxazoles; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D008274:Magnesium; D016257:Fura-2