PMID:9693004
| Citation |
Jung, H, Tebbe, S, Schmid, R and Jung, K (1998) Unidirectional reconstitution and characterization of purified Na+/proline transporter of Escherichia coli. Biochemistry 37:11083-8 |
|---|---|
| Abstract |
A simple approach for large-scale purification and unidirectional reconstitution of the Na+/proline transporter of Escherichia coli (PutP) is described. The procedure is based on the insertion of a highly polar peptide composed of 17 amino acids including a 6His tag at the C-terminus of the transporter. Purification of the hybrid protein is achieved by Ni+-NTA affinity (purity >95%) and ion exchange chromatography (purity >99%). The purified transporter is reconstituted into preformed, detergent-destabilized liposomes. Detergent is removed slowly by adsorption to polystyrene beads. The highest activities [Vmax = 1.1 x 10(3) nmol min-1 (mg of protein)-1] are measured when Triton X100 is used for liposomes destabilization at a concentration corresponding to the onset of lipid solubilization. Site-directed labeling of PutP and site-specific proteolytic cleavage indicate that the transporter is inserted into proteoliposomes in an inside-out orientation. Reconstituted PutP is able to accumulate proline against a concentration gradient in the presence of an inwardly directed electrochemical Na+ or Li+ gradient, while a pH gradient does not affect transport. The apparent proline affinity of PutP in proteoliposomes is similar to the value determined with intact cells. Interestingly however, the apparent Na+ affinity of reconstituted PutP is reduced by a factor of about 25 compared to cells, suggesting a lower cation affinity on the cytosolic side of PutP relative to the outside. |
| Links |
PubMed Online version:10.1021/bi980684b |
| Keywords |
Amino Acid Transport Systems, Neutral; Biological Transport; Chromatography, Affinity; Chromatography, Ion Exchange; Detergents; Escherichia coli/enzymology; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/isolation & purification; Membrane Transport Proteins/metabolism; Nephelometry and Turbidimetry; Octoxynol; Proline/metabolism; Proteolipids/metabolism; Sodium/metabolism; Solubility |
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