Surface potential in rat liver mitochondria: terbium ion as a phosphorescent probe for surface potential

Abstract

The binding and phosphorescence of Tb3+ in rat liver mitochondria and submitochondrial particles were investigated. Mitochondria were treated briefly with N-ethylmaleimide (NEM) to prevent phosphate leak and Tb3+ chelation. Up to 30 nmol of Tb3+/mg of protein binds to mitochondrial membranes with high apparent affinity (Kd .simeq. 6 .mu.M). Generation of a membrane potential had no significant effect on the apparent affinity or capacity of Tb3+ binding in NEM-treated mitochondria. Mitochondrial bound Tb3+ phosphorescence can be induced selectively by excitation of aromatic amino acid residues. The decay of mitochondrial bound Tb3+ phosphorescence is biphasic. The phosphorescence of the slow phase (t1/2 [half-time] = 0.45-0.70 ms) is quenched by monovalent salts, indicating a negative surface potential at low salt medium of -5.4 .+-. 2.8 mV [10 mM 3-(N-morpholino)-propanesulfonic acid, pH 7.2, 5 .mu.M Tb3+]. In submitochondrial particles, a surface potential of -6.5 .+-. 2.7 mV was estimated under the same conditions. Energization did not affect the surface potential significantly in submitochondrial particles and only slightly in mitochondrial particles and only slightly in mitochondria. Analysis of the phosphorescence of mitochondrial bound Tb3+ reveals 2 binding sites with high (Kd = 1.5 .mu.M) and low affinity (Kd = 29 .mu.M). The high-affinity site is tentatively identified as the Ca2+ carrier. A fraction of the carrier-bound Tb3+ phosphorescence decays rapidly, presumably as a result of energy transfer to cytochromes in the membrane core. These intramembrane sites appear to move to the surface on the generation of a membrane potential. The salt effect on the phosphorescence of the slow phase may serve as a reliable measure of delocalized surface potential in mitochondria and submitochondrial particles. Tb3+ binding to the high-affinity site may be useful as a probe for the mitochondrial Ca2+ translocator.