The hOCTN1 (human organic cation transporter 1) overexpressed in Escherichia coil 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 [(14)C]tetraethylammonium, [(3)H]carnitine or [(3)H]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. [(14)C]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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|Titolo:||Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter overexpressed in Escherichia coli|
|Data di pubblicazione:||2011|
|Appare nelle tipologie:||1.1 Articolo in rivista|