Hepatic mitochondrial transport of glutathione: Studies in isolated rat liver mitochondria and H4IIE rat hepatoma cells☆
Section snippets
Materials
GSH, iodoacetic acid, 1-fluoro-2, 4-dinitrobenzene, antimycin A (AA), phosphoenolpyruvate (PEP), oligomycin, and atractyloside were purchased from Sigma Chemical Co. (St. Louis, MO). Phenylsuccinate and butylmalonate were purchased from Aldrich Chemical Co. (Milwaukee, WI). l-[3H-Glycyl]-GSH (44.8 Ci/mmol) and [14C]-2-OG (281 mCi/mmol) was purchased from DuPont NEN (Boston, MA).
Preparation of mitochondria
Male Sprague–Dawley rats (175–225 g) were purchased from Harlan (Indianapolis, IN). Rats were housed in cages in the
Kinetics of GSH uptake
The time course for uptake of 5 mM GSH in liver mitochondria was measured at 25 °C by the centrifugation–resuspension method (Fig. 1A). Initial mitochondrial GSH content was about 0.6 nmol/mg protein. In the presence of 7.5 mM extramitochondrial GSH, the intramitochondrial content of GSH increased rapidly within 5 min to an equilibrium level of 2.2 nmol/mg protein. GSH uptake was concentration dependent (Fig. 1B) and was uniphasic (Fig. 2), exhibiting a Km of 4.08 mM and a Vmax of 3.06 nmol/mg protein
Discussion
In the present study, we demonstrate that both the DIC and OGC contribute to, but are not entirely responsible for, the mitochondrial uptake of GSH in rat liver. Unlike our previous findings in rat kidney mitochondria [3], [4], where we found that function of these two inner membrane carriers could account for nearly all of the observed uptake of GSH, in hepatic mitochondria the DIC and OGC only appear to account for approximately 50% of the observable transport. We further demonstrated the
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This research was supported by NIH Grant R01-DK40725 to L.H.L. and by the NIEHS Center for Molecular Toxicology with Human Applications (Grant P30-ES06639) at Wayne State University.