FIGURE 2.

Recording of changes in matrix volumes by light scattering of isolated rat heart mitochondria during transitions to solution with respiratory substrates. Effects of nigericin and calcium. Isolated mitochondria were added into the fluorimetric chamber in the presence of substrates. (A) Mitochondria isolated in a sucrose medium (250 mM) without respiratory substrates (see Materials and Methods) were added into solution B with glutamate and malate as respiratory substrates and with K⁺, whose entry by K⁺-channel (open in absence of ATP) induces swelling, which is observed as a decrease of the intensity of reflected light. Addition of Ca²⁺ (5 mM, 2 times) induced maximal mitochondrial swelling due to opening of the permeability transition pore, which is seen as a decrease of optical density at 520–520 nm. (B) The same as A, in solution B addition of nigericin (in absence of bovine serum albumin to avoid binding of nigericin) resulted in some contraction of matrix of mitochondria due to K⁺–H⁺ exchange (Nicholls and Ferguson, 2002). (C) Isolated mitochondria were introduced into the spectrofluorimeter chamber with solution B with glutamate and malate as substrates, and the Rh 123 fluorescence was recorded as described in Materials and Methods. Normal membrane potential was seen in solution B. Two successive additions of 5 mM Ca²⁺ led to a complete depolarization of the mitochondrial membrane, accompanied with the complete collapse of membrane potential and release of Rh 123 from mitochondrial matrix into the medium. (D) The dependence of respiration rate upon the ADP concentration in Lineweaver-Burk plots in sucrose solution and solution B without and with nigericin. Both in sucrose solution and solution B the apparent K_m for ADP was equal to 8 μM. Nigericin induced the parallel shift of a straight line, giving both lower K_m and V_m.