Measurement of matrix free Mg2+ concentration in rat heart mitochondria by using entrapped fluorescent probes.
1. The concentration of free Mg2+ ([Mg2+]m) within the matrix of isolated rat heart mitochondria was measured after loading of the mitochondria with the fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2. No detectable change in total mitochondrial magnesium content occurred during loading with the indicators. Apparent Kd values for Mg2+ of 3.7 mM and 2.3 mM were obtained for mag-indo-1 and mag-fura-2 respectively within mitochondria permeabilized to bivalent cations with ionomycin and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These values are 2.7- and 1.8-fold greater respectively than those obtained for the free acid forms of the dyes in incubation medium. 2. Based on the above Kd values, mitochondrial matrix Mg2+ concentrations were found to lie in the range 0.8-1.5 mM in the absence, or immediately after the addition, of a respiratory substrate. 3. Incubation of mitochondria in the presence of respiratory substrate, but in the absence of external Mg2+, led to a time-dependent decline in [Mg2+]m to about half the initial values after 5 min. This was accompanied by a fall in the total mitochondrial magnesium content from 12.7 to 7.0 nmol/mg of protein. 4. ADP (0.5 mM), ATP (0.5 mM) or 10 mM-NaCl had no significant effect on the fall in [Mg2+], whereas 1 microM-nigericin blocked, and 0.3 microM-valinomycin accelerated, the fall. 5. External Mg2+ concentrations above 1 mM progressively inhibited and reversed the decline in free and total mitochondrial Mg2+.
Rutter
G A
GA
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Osbaldeston
N J
NJ
McCormack
J G
JG
Denton
R M
RM
D000818:Animals; D005456:Fluorescent Dyes / Q000032:analysis*; D016257:Fura-2 / Q000378:metabolism; D006868:Hydrolysis; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000032:analysis* / Q000378:metabolism; D008929:Mitochondria, Heart / Q000737:chemistry*; D051381:Rats; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007211:Indoles; D008274:Magnesium; C048960:indo-1; D016257:Fura-2