Journal
BIOCHEMICAL JOURNAL
Volume 439, Issue -, Pages 227-233Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20110544
Keywords
liposome; OCTN1; reconstitution; tetraethylammonium; transport
Categories
Funding
- Ministero dell'Universita e della Ricerca [2006054479]
- University of Calabria progetti
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The hOCTN1 (human organic cation transporter 1) overexpressed in Escherichia coli and purified by Ni-chelating chromatography has been reconstituted in liposomes by detergent removal with a batch-wise procedure. The reconstitution was optimized with respect: to the protein concentration, the detergent/phospholipid ratio and the time of incubation with Amber lite XAD-4 resin. Time-dependent [C-14]tetraethylammonium, [H-3]carnitine or [H-3]ergothioneine uptake was measured in proteoliposomes with activities ratios of 8:1.3:1 respectively. Optimal activity was found at pH 8.0. The transport depended on intraliposomal ATP. [C-14]tetraethylammonium transport was inhibited by several compounds. The most effective were acetylcholine and gamma-butyrobetaine, followed by acetylcarnitine and tetramethylammonium. Reagents such as pyridoxal 5-phosphate, MTSES [sodium (2-sulfonatoethyl) methanethiosulfonate] and mercurials strongly inhibited the transport. From kinetic analysis of tetraethylammonium transport a K-m of 0.77 mM was calculated. Acetylcholine and gamma-butyrobetaine behaved as competitive inhibitors of TEA (tetraethylammonium) transport with K-i values of 0.44 and 0.63 mM respectively.
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