Abstract
The dynamics and environment of water in suspensions of isolated rat liver mitochondria have been investigated by 1H NMR. NMR longitudinal and transversal relaxation times (T1 and T2) were measured in the resuspension medium (2.65 s and 44.57 ms) and in mitochondrial suspensions (1.74 s and 23.14 s), respectively. Results showed monoexponential relaxation in both cases, suggesting a fast water exchange across the inner mitochondrial membrane. Ferromagnetically induced shift of the extramitochondrial water with nonpermeant ferromagnetic particles revealed no detectable water signal from the intramitochondrial compartment, confirming the fast exchange case. Simulations on a two-compartment model indicated that the intramitochondrial water residence time has an upper limit of approximately 100 microseconds. Calculated intramitochondrial relaxation times revealed that the intramitochondrial environment has an apparent viscosity 30 times larger than the resuspension medium and 15 times larger than the cytosol of erythrocytes. The higher apparent viscosity of the mitochondrial matrix could account for reductions of more than one order of magnitude in the diffusion coefficient of water and other substrates, limitations in the rate of enzymatic reactions which are diffusion controlled and a more favorable formation of multienzyme complexes.
Highlights
Adequate understanding of the dynamics of water at the cellular level involves the determination of the water exchange rate across the different intracellular membranes and the study of the water environment within the different organelles [1,2,3,4]
Our methodology is based on comparisons of the 1H NMR longitudinal (T1)2 and transversal (T2) relaxation times of the water protons in the mitochondrial suspensions with those observed in the resuspension medium without mitochondria
T1 (T2) measurements of the resuspension medium (n ϭ 6) and of the mitochondrial suspensions (n ϭ 5) were always monoexponential with values of 2.65 Ϯ 0.2 s (44.57 Ϯ 2.2 ms) and 1.74 Ϯ 0.1 s (23.14 Ϯ 1.6 ms), respectively. These results indicate that the relaxation times of intramitochondrial water are significantly shorter than those of water in the resuspension medium
Summary
Adequate understanding of the dynamics of water at the cellular level involves the determination of the water exchange rate across the different intracellular membranes and the study of the water environment within the different organelles [1,2,3,4]. The transport of water across the inner mitochondrial membrane and the environmental properties of the intramitochondrial space remain poorly understood. Our methodology is based on comparisons of the 1H NMR longitudinal (T1) and transversal (T2) relaxation times of the water protons in the mitochondrial suspensions with those observed in the resuspension medium without mitochondria. This approach is specially suited for the noninvasive study of the water environment in biological systems since the magnetic relaxation properties of the water protons are a direct consequence of their translational and rotational motions
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