Table 1.
RLIP76 protein and transport activity in human kidney normal (mesangial) and cancer (Caki-2) cells
| RLIP76 protein |
Transport activity (pmol/min/mg) |
|||
|---|---|---|---|---|
| μg/108 | Total crude protein (%) | 14C-doxorubicin | 3H-DNP-SG | |
| Mesangial (normal) | 17 ± 2 | 0.22 | 43 ± 6 | 138 ± 14 |
| Caki-2 (malignant) | 46 ± 3 | 0.62 | 135 ± 19 | 716 ± 64 |
NOTE: Cell lines were cultured in respective medium and homogenate was prepared from 100 × 106 cells. RLIP76 was purified from total crude membrane fraction using DNP-SG affinity column chromatography and quantified by ELISA. SDS-PAGE and Western blot of purified RLIP76 are presented in Fig. 1B and C, respectively. For transport studies, IOVs prepared from 20 × 106 cells were enriched for IOVs by wheat germ agglutinin affinity chromatography (24). Transport activity was calculated from measurements of uptake of 14C-doxorubicin (specific activity, 8.5 × 104 cpm/nmol) and 3H-DNP-SG (specific activity, 3.6 × 103 cpm/nmol) into the IOVs (20 μg/30 μL reaction mixture) in the presence or absence of 4 mmol/L ATP as described (24). Each transport experiment was done in triplicates in three separate experiments (n = 9).