Novel properties of hepatic canalicular reduced glutathione transport revealed by radiation inactivation.
Transport of GSH at the canalicular pole of hepatocytes occurs by a facilitative carrier and can account for approximately 50% of total hepatocyte GSH efflux. A low-affinity unit with sigmoidal kinetics accounts for 90% of canalicular transport at physiological GSH concentrations. A low-capacity transporter with high affinity for GSH has also been reported. It is not known whether the same or different proteins mediate low- and high-affinity GSH transport, although they do differ in inhibitor specificity. The bile of rats with a mutation in the canalicular multispecific organic anion transporter (cMOAT or MRP-2, a 170-kDa protein) is deficient in GSH, implying that cMOAT may transport GSH. However, transport of GSH in canalicular membrane vesicles (CMV) from these mutant rats remains intact. We examined the functional size of the two kinetic components of GSH transport by radiation inactivation of GSH uptake in rat hepatic CMV. High-affinity transport of GSH was inactivated as a single exponential function of radiation dose, yielding a functional size of approximately 70 kDa. In contrast, low-affinity canalicular GSH transport exhibited a complex biexponential response to irradiation, characterized by an initial increase followed by a decrease in GSH transport. Inactivation analysis yielded a approximately 76-kDa size for the low-affinity transporter. The complex inactivation indicated that the low-affinity transporter is associated with a larger protein of approximately 141 kDa, which masked approximately 80% of the potential transport activity in CMV. Additional studies, using inactivation of leukotriene C4 transport, yielded a functional size of approximately 302 kDa for cMOAT, indicating that it functions as a dimer.
Mittur
A V
AV
Department of Medicine, University of Southern California, Los Angeles 90033, USA.
Kaplowitz
N
N
Kempner
E S
ES
Ookhtens
M
M
D015720:5'-Nucleotidase / Q000378:metabolism; D000818:Animals; D027321:Anion Transport Proteins; D001648:Bile Canaliculi / Q000378:metabolism* / Q000528:radiation effects; D001692:Biological Transport / Q000502:physiology / Q000528:radiation effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000528:radiation effects; D004789:Enzyme Activation / Q000528:radiation effects; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D015720:5'-Nucleotidase; D005978:Glutathione