Transmembrane gradients of free Na+ in isolated heart mitochondria estimated using a fluorescent probe.
The concentration of free Na+ in the matrix of isolated pig heart mitochondria has been monitored using the fluorescent probe sodium-binding benzofuran isophthalate (SBFI) developed by Minta and Tsien (J. Biol. Chem. 264: 19449-19457, 1989). SBFI was sequestered in the matrix by hydrolysis of the permeant acetoxymethyl ester. The sequestered probe showed altered quantum efficiency and excitation spectra in the presence and absence of Na+ when compared with SBFI free acid in solution. Fluorescence was calibrated in situ by using ionophores to equilibrate matrix [Na+] with external [Na+]. SBFI fluorescence showed that matrix [Na+] increased linearly as external [Na+] was increased to 95 mM in the presence or absence of respiration. Respiring mitochondria maintained a Na+ gradient (Na+ out greater than Na+in) of approximately 8.0. The corresponding gradient in nonrespiring mitochondria was approximately 2.0. The Na+ gradient was nearly equivalent to the H+ gradient in the presence or absence of respiration. The uptake of Pi by respiring mitochondria decreased matrix pH and increased matrix [Na+]. It is concluded that isolated mitochondria maintain a Na+ gradient across the inner membrane as a result of the activity of the endogenous Na(+)-H+ antiport.
Jung
D W
DW
Department of Medical Biochemistry, Ohio State University Medical Center, Columbus 43210.
Apel
L M
LM
Brierley
G P
GP
D000818:Animals; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008566:Membranes / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism*; D009994:Osmolar Concentration; D012964:Sodium / Q000378:metabolism*; D013552:Swine
D005452:Fluoresceins; D005456:Fluorescent Dyes; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium