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.
Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter overexpressed in Escherichia coli
Pochini Lorena;Scalise Mariafrancesca;Galluccio Michele;Indiveri C
2011-01-01
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
