Development and Characterization of Mammalian Cells Derived P-gp, BCRP, MRP2 and BSEP Membrane Vesicles

2016 ◽  
Author(s):  
Na Li
Keyword(s):  
Mammalian Cells ◽  
Membrane Vesicles

scholarly journals Characterization of the plasma-membrane calcium pump from Trypanosoma cruzi

1995 ◽  
Vol 306 (1) ◽  
pp. 299-303 ◽  
Author(s):  
G Benaim ◽  
S N J Moreno ◽  
G Hutchinson ◽  
V Cervino ◽  
T Hermoso ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Membrane Vesicles ◽  
High Sensitivity ◽  
Bovine Brain ◽  
Calcium Pump ◽  
Plasma Membrane Vesicles ◽  
P Type

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma-membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps.

Expression and characterization of a functional rat liver Na+ bile acid cotransport system in COS-7 cells

1994 ◽  
Vol 266 (3) ◽  
pp. G382-G387 ◽  
Author(s):  
J. L. Boyer ◽  
O. C. Ng ◽  
M. Ananthanarayanan ◽  
A. F. Hofmann ◽  
C. D. Schteingart ◽  
...  
Keyword(s):  
Bile Acid ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Membrane Vesicles ◽  
Kinetic Studies ◽  
Rat Hepatocytes ◽  
Cos Cells ◽  
Bile Acid Transporter ◽  
Cotransport System

A cDNA for the rat liver sodium-dependent bile acid cotransporter was expressed in COS-7 cells to study the functional properties of the translated protein in a mammalian cell line. A 1.2-kb insert was ligated into a pMAMneo vector and transiently transfected using electroporation. After optimal conditions were established, the transiently transfected COS cells were screened with fluorescent-conjugated labeled bile acids for evidence of expression of the cotransporter after 48 h. The uptake of [3H]taurocholate ([3H]TC) was then determined in cells transfected with or without the bile acid insert. Progressive uptake of [3H]TC (0.45 microM) was observed for 30 min in the presence of sodium. In contrast, no uptake of [3H]TC was observed in the absence of sodium, in nontransfected COS cells, or in COS cells transfected with the empty plasmid. Kinetic studies revealed a Michaelis constant (Km) of 29 microM, essentially identical to the Km of this cotransporter described in intact rat hepatocytes and membrane vesicles. Uptake of [3H]TC (5.0 microM) at 5 min (n = 3-6) was inhibited by 100 microM taurochenodeoxycholic acid (81%), tauroursodeoxycholic acid (77%), cholic acid (55%), chenodeoxycholic acid (74%), and ursodeoxycholic acid (56%) but not by 100 microM taurodehydrocholate, 1 mM probenecid, or 100 microM bilirubin. In contrast, bumetanide (500 microM) inhibited [3H]TC uptake by 52%. These studies indicate that the isolated cDNA codes for a physiological bile acid transporter present in rat hepatocytes and that posttranslational factors present in mammalian cells may not be as important in defining properties of this cotransport system.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

scholarly journals Purification and characterization of functional recombinant alpha-amidating enzyme secreted from mammalian cells.

1990 ◽  
Vol 265 (29) ◽  
pp. 17694-17699 ◽  
Author(s):  
G A Beaudry ◽  
N M Mehta ◽  
M L Ray ◽  
A H Bertelsen
Keyword(s):  
Mammalian Cells ◽  
Purification And Characterization

scholarly journals Expression and characterization of human antithrombin III synthesized in mammalian cells.

1987 ◽  
Vol 262 (30) ◽  
pp. 14766-14772 ◽  
Author(s):  
L C Wasley ◽  
D H Atha ◽  
K A Bauer ◽  
R J Kaufman
Keyword(s):  
Mammalian Cells ◽  
Antithrombin Iii

scholarly journals Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells.

1988 ◽  
Vol 263 (22) ◽  
pp. 10817-10823
Author(s):  
H V Le ◽  
L Ramanathan ◽  
J E Labdon ◽  
C A Mays-Ichinco ◽  
R Syto ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interleukin 4 ◽  
Isolation And Characterization ◽  
Multiple Variants
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