Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris--functional and kinetic asymmetry revealed in proteoliposomes.
作者： Piero Pingitore , Lorena Pochini , Mariafrancesca Scalise , Michele Galluccio , Kristina Hedfalk
DOI: 10.1016/J.BBAMEM.2013.05.034
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摘要: Abstract The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni2 +-chelating gel filtration chromatography. protein reconstituted liposomes detergent removal with a batch-wise procedure. Time dependent [3H]glutamine/glutamine antiport measured proteoliposomes which active only the presence of external Na+. Internal Na+ slightly stimulated antiport. Optimal activity found at pH 7.0. A substantial inhibition transport observed Cys, Thr, Ser, Ala, Asn Met (≥ 70%) mercurials methanethiosulfonates (≥ 80%). Heterologous [3H]glutamine other neutral acids also studied. showed asymmetric specificity for acids: Val, were inwardly transported, while Gln, Asn, Thr transported bi-directionally. From kinetic analysis Km values 0.097 1.8 mM on internal sides proteoliposomes, respectively. side 32 mM. homology structural model hASCT2 built using GltPh Pyrococcus horikoshii as template. Cys395 Cys residue externally exposed, thus being potential target SH reagents and, hence, potentially involved mechanism.
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