Download presentation
Presentation is loading. Please wait.
1
Volume 123, Issue 5, Pages 1649-1658 (November 2002)
The human bile salt export pump: Characterization of substrate specificity and identification of inhibitors Jane A. Byrne, Sandra S. Strautnieks, Giorgina Mieli–Vergani, Christopher F. Higgins, Kenneth J. Linton, Richard J. Thompson Gastroenterology Volume 123, Issue 5, Pages (November 2002) DOI: /gast Copyright © 2002 American Gastroenterological Association Terms and Conditions
2
Fig. 1 Strategy to generate a full-length ABCB11 cDNA. ABCB11 cDNA fragments 1–3 were amplified individually and restriction endonuclease sites introduced at the 5' and 3' ends to allow their cloning are shown in bold. Unique restriction endonuclease sites in the ABCB11 sequence, used to reconstruct the full-length cDNA from the cloned individual fragments, are shown in Roman type. Codons for a 6 histidine tag (his tag) were introduced to the 3' end of fragment 3 to generate fragment 3-plus-histidine. Gastroenterology , DOI: ( /gast ) Copyright © 2002 American Gastroenterological Association Terms and Conditions
3
Fig. 2 Western blot analysis of BSEP protein expression and optimization of MOI in High Five cell membranes. Lanes were loaded with 30 μg membrane protein prepared from uninfected High Five cells (neg), parental virus–infected High Five cells (mock), and High Five cells infected with the ABCB11 baculovirus at MOI of 4, 7.5, 10, and 15, 3 days after infection. Western blotting was performed with an antihistidine-tag antibody (see Materials and Methods). Gastroenterology , DOI: ( /gast ) Copyright © 2002 American Gastroenterological Association Terms and Conditions
4
Fig. 3 ATPase activity of BSEP in High Five cell membranes. Basal ATPase activity was measured in membrane vesicles prepared from uninfected High Five cells (neg), parental virus–infected High Five cells (mock), and cells infected with the ABCB11 baculovirus (BSEP) in the presence and absence of 200 μmol/L vanadate. Results represent the mean of 3 separate experiments from 2 vesicle preparations. Gastroenterology , DOI: ( /gast ) Copyright © 2002 American Gastroenterological Association Terms and Conditions
5
Fig. 4 ATP-dependent taurocholate uptake by BSEP. Membrane vesicles prepared from High Five cells infected with the ABCB11 baculovirus were used to determine initial (30 seconds) ATP-dependent uptake of increasing concentrations of [3H]-taurocholate. Kinetic parameters were fitted to uptake data by using the Michaelis-Menten equation. Results represent the mean of 3 separate experiments from 2 vesicle preparations. Gastroenterology , DOI: ( /gast ) Copyright © 2002 American Gastroenterological Association Terms and Conditions
6
Fig. 5 Specificity of BSEP for various bile salts. ATP-dependent uptake of 3 μmol/L, 6 μmol/L, and 9 μmol/L [3H]-taurocholate was measured in the presence of various concentrations of indicated bile salts. Dixon plot analysis was used to evaluate the kinetics of the bile salt-induced inhibition of ATP-dependent taurocholate uptake. Results represent the mean of 3 separate experiments from 2 vesicle preparations. Gastroenterology , DOI: ( /gast ) Copyright © 2002 American Gastroenterological Association Terms and Conditions
7
Fig. 6 Inhibition of taurocholate uptake by drugs. ATP-dependent uptake of 3 μmol/L, 4.5 μmol/L, and 6 μmol/L [3H]-taurocholate was measured in the presence of various concentrations of indicated drugs. Dixon plot analysis was used to evaluate the kinetics of the drug-induced inhibition of ATP-dependent taurocholate uptake. Results represent the mean of 3 separate experiments from 2 vesicle preparations. Gastroenterology , DOI: ( /gast ) Copyright © 2002 American Gastroenterological Association Terms and Conditions
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.