Figure 5.

This figure presents information about the transport mechanism and kinetics of chimpanzee SLC22A10.

A. The uptake of seven steroid glucuronides and two steroids in HEK293 cells stably transfected with chimpanzee SLC22A10 (552 amino acids) was measured using LC/MS-MS to determine the accumulation of the compounds.

B. The effect of pH on accumulation of [³H]-estradiol-17b-glucuronide in HEK293 cells stably transfected with chimpanzee SLC22A10 isoforms expressing 533 and 552 amino acids was investigated.

C. The effect of sodium and chloride on accumulation of [³H]-estradiol-17b-glucuronide in HEK293 cells stably transfected with 533 and 552 amino acid isoforms of chimpanzee SLC22A10 was investigated.

D. The effects of trans-stimulation of [³H]-estradiol-17b-glucuronide uptake by chimpanzee SLC22A10 was determined. Uptake was trans-stimulated by preloading the cells with 2 mM of butyrate, glutaric acid, alpha-ketoglutarate, or succinic acid for 2 hours, and then measuring the uptake of [³H]-estradiol-17b-glucuronide after 15 minutes. The data are presented as mean ± S.D. and were normalized by setting the uptake of SLC22A10-expressing cells trans-stimulated by HBSS to 1.0. Trans-stimulation of [³H]-estradiol-17b-glucuronide by glutaric acid was observed for both isoforms of chimpanzee SLC22A10. E. The kinetics of [³H]-estradiol-17b-glucuronide uptake for chimpanzee SLC22A10 isoforms expressing 533 and 552 amino acids were analyzed. The uptake rate was evaluated at 5 minutes and the data were fit to a Michaelis-Menten equation. To fit the kinetic curve to a Michaelis-Menten equation, the concentration of estradiol-17b-glucuronide is set up to 10 μM. The figure shows a representative plot from one experiment. All experiments were repeated once, in triplicate and showed similar results.