Preparation of plasma membrane and the analysis of transport kinetics and driving forces
Niinuma, K.; Suzuki, H.; Yamazaki, M.; Sugiyama, Y.
Nihon Rinsho. Japanese Journal of Clinical Medicine 54(3): 692-703
1996
ISSN/ISBN: 0047-1852 PMID: 8904225 Document Number: 456602
The principles of isolating plasma membrane (basolateral membrane and brush border membrane), and separating right side-out and inside-out membrane vesicles were described. We then focused on the liver plasma membrane. Liver plasma membrane vesicles have been used to investigate the mechanism for the hepatobiliary transport of ligands. We precisely describe our protocol to isolate the sinusoidal (SMV) and canalicular membrane vesicles (CMV) from rat liver using the percoll gradient. Methods to determine the purity and orientation of SMV and CMV are also mentioned. Furthermore, procedures for the transport studies with SMV and CMV are provided; we refer to the electrogenic sodium dependent transport of taurocholic acid and alanine into SMV as an example to characterize the secondary active transport system. ATP-dependent uptake of S-(2,4-dinitrophenyl)-glutathione (DNP-SG) into CMV is also cited as an example to clarify the primary active transport mechanism. Kinetic analysis for the ATP-dependent uptake of [3H] DNP-SG into CMV is also described.