Type | DTT | High Affinity | Low Affinity | ||
---|---|---|---|---|---|
Km | Vmax | Km | Vmax | ||
mM | nmol/mg protein/10 s | mM | nmol/mg protein/10 s | ||
bLPM IO | − | 0.058 ± 0.064 | 0.089 ± 0.099 | 13.2 ± 3.84-150 | 18.4 ± 3.0 |
bLPM RO | − | 0.129 ± 0.231 | 0.054 ± 0.101 | 13.2 ± 3.84-150 | 9.69 ± 1.9 |
HeLa Control | − | N.D. | N.D. | 15.3 ± 3.6 | 23.8 ± 4.2 |
+ | N.D. | N.D. | 13.0 ± 2.4 | 13.1 ± 2.7 | |
HeLa Oatp1 | − | N.D. | N.D. | 19.8 ± 2.7 | 42.1 ± 4.2 |
+ | N.D. | N.D. | 17.8 ± 4.7 | 32.7 ± 4.9 | |
adj. | N.D. | N.D. | 15.9 ± 1.9 | 32.2 ± 2.7 |
Initial rates (10 s) were determined from uptake of [35S]GSH at various concentrations ranging from 0.0002 μM to 50 mM in membrane vesicles under voltage-clamped conditions. Kinetic parameters of GSH uptake in untreated and DTT-pretreated membrane vesicles were estimated by curve fitting using a single Michaelis-Menten component or a combination of a Michaelis-Menten and a sigmoid Hill component as described under Materials and Methods. Values in control and Oatp1-expressing HeLa membrane vesicles represent mean ± S.E. (n = 3 to 5); values in IO and RO bLPM represent mean ± S.E. (n = 4 to 5).
N.D., none detected; adj., the fraction of GSH uptake inhibited by DTT in control vesicles was subtracted from uptake in untreated Oatp1 vesicles.
↵4-150 , sigmoid component with Hill coefficient > 1.2.