title: Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter overexpressed in Escherichia coli.
abstract: 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 Amberlite 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 acetyl-choline and γ-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 γ-butyrobetaine behaved as competitive inhibitors of TEA (tetraethylammonium) transport with K(i) values of 0.44 and 0.63 mM respectively.
authors: Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende, Italy.
authors: Lorena
authors: L
authors: Pochini
authors: Mariafrancesca
authors: M
authors: Scalise
authors: Michele
authors: M
authors: Galluccio
authors: Linda
authors: L
authors: Amelio
authors: Cesare
authors: C
authors: Indiveri
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D027981:Symporters; D019789:Tetraethylammonium / Q000378:metabolism
chemical_list: D008081:Liposomes; D027701:Organic Cation Transport Proteins; D011994:Recombinant Proteins; C109807:SLC22A4 protein, human; D027981:Symporters; D019789:Tetraethylammonium