Radiation inactivation studies of hepatic sinusoidal reduced glutathione transport system

Abstract

Sinusoidal transport of reduced glutathione (GSH) is a carrier-mediated process. Perfused liver and isolated hepatocyte models revealed a low-affinity transporter with sigmoidal kinetics (K(m) approximately 3.2-12 mM), while studies with sinusoidal membrane vesicles (SMV) revealed a high-affinity unit (K(m) approximately 0.34 mM) besides a low-affinity one (K(m) approximately 3.5-7 mM). However, in SMV, both the high- and low-affinity units manifested Michaelis-Menten kinetics of GSH transport. We have now established the sigmoidicity of the low-affinity unit (K(m) approximately 9) in SMV, consistent with other models, while the high-affinity unit has been retained intact with Michaelis-Menten kinetics (K(m) approximately 0.13 mM). We capitalized on the negligible cross-contributions of the two units to total transport at the low and high ends of GSH concentrations and investigated their characteristics separately, using radiation inactivation, as we did in canalicular GSH transport (Am. J. Physiol. 274 (1998) G923-G930). We studied the functional sizes of the proteins that mediate high- and low-affinity GSH transport in SMV by inactivation of transport at low (trace and 0.02 mM) and high (25 and 50 mM) concentrations of GSH. The low-affinity unit in SMV was much less affected by radiation than in canalicular membrane vesicles (CMV). The target size of the low-affinity sinusoidal GSH transporter appeared to be considerably smaller than both the canalicular low- and high-affinity transporters. The high-affinity unit in SMV was markedly inactivated upon irradiation, revealing a single protein structure with a functional size of approximately 70 kDa. This size is indistinguishable from that of the high-affinity GSH transporter in CMV reported earlier.

The Thiol Sensitivity of Glutathione Transport in Sidedness-Sorted Basolateral Liver Plasma Membrane and in Oatp1-Expressing HeLa Cell Membrane

“…Membrane fractions were stored at Ϫ80°C in buffer A (20 mM KCl, 240 mM sucrose, 0.2 mM CaCl 2 , 0.2 mM MgCl 2 , 5 mM KHCO 3 , and 10 mM HEPES/Tris, pH 7.4). The purity and enrichment of various enzymes, including 5Ј-nucleotidase, in membrane vesicles were determined by assaying their total activity in the presence of 0.1% Triton X-100 (Mittur et al, 2000).…”

“…The expression and activity of Oatp1 was determined by comparing Na ϩ -independent uptake of taurocholate (10 M) in membrane vesicles from control and Zn 2ϩ -induced HeLa cells. Uptake of alanine, taurocholate, and lglutamate were determined as described previously (Meier et al, 1984a;Mittur et al, 2000 Uptake and Kinetics of Substrates in Membrane Vesicles. The uptake of radiolabeled substrates in vesicles was determined by rapid filtration (Kannan et al, 1999;Mittur et al, 2000).…”

“…Uptake of alanine, taurocholate, and lglutamate were determined as described previously (Meier et al, 1984a;Mittur et al, 2000 Uptake and Kinetics of Substrates in Membrane Vesicles. The uptake of radiolabeled substrates in vesicles was determined by rapid filtration (Kannan et al, 1999;Mittur et al, 2000). In each preparation and at all substrate concentrations, measurements were made in duplicate and the mean values of the duplicate determinations were used.…”

“…Sinusoidal efflux is critical in interorgan homeostasis of GSH and thioldisulfides (Ookhtens and Kaplowitz, 1998) and presumably in regulation of redox status in plasma. Carrier-mediated transport of GSH across the sinusoidal membrane of hepatocytes is mediated by high-and low-affinity components (Inoue et al, 1984;Ookhtens et al, 1985;Aw et al, 1986;Mittur et al, 2000). GSH transport in perfused livers, hepatocytes, and basolateral plasma membrane vesicles (bLPM) is mediated by a low-affinity transporter with sigmoid kinetics (K m ϳ3-12 mM; n H ϳ2-3).…”

“…GSH transport in perfused livers, hepatocytes, and basolateral plasma membrane vesicles (bLPM) is mediated by a low-affinity transporter with sigmoid kinetics (K m ϳ3-12 mM; n H ϳ2-3). The role of the high-affinity component (K m ϳ150 M) in regulation of sinusoidal GSH efflux is not clear because it has a 10-fold lower capacity than the low-affinity component and is saturated at normal cellular levels of GSH (Aw et al, 1986;Mittur et al, 2000). A functional size of ϳ70 kDa has been estimated for the highaffinity component observed in bLPM (Mittur et al, 2000).…”