Abstract
The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.
Highlights
This report describes the overexpression, purification, and quaternary structure of AdiC, along with an initial description of its membrane transport behavior
In E. coli cells, AdiC-mediated Arg-Agm exchange across the inner membrane is undetectable at neutral pH but is acutely activated by acid challenge (4 – 6)
This does not mean that AdiC itself is acid-activated since in cellular conditions, transport is strictly coupled to Agm generation by arginine decarboxylase, an acid-activated enzyme [26]
Summary
Expression, Purification, and Reconstitution—The coding sequence of the adiC gene of E. coli was inserted into the XbaI/ HindIII cassette of vector pASK-IBA2 [11] behind a ribosomebinding sequence (TAACGAGGGCAAAAA), as described for a ClϪ transporter [12]. After concentrating to 0.5–1 ml, the sample was treated with thrombin (Roche Applied Science, 0.1 units/mg of AdiC) at 4 °C overnight, and AdiC was purified on a Superdex 200 size exclusion column in WB with 5 mM DM This column was calibrated [13] with the elution times in DM solutions of functionally active integral membrane protein complexes of known molecular sizes (and roughly similar non-pathological shapes) as follows: MthK, 250 kDa [14], 10.5 ml; CLC-ec1-FAB complex [15], 200 kDa, 11.1 ml; MloK1 [16], 150 kDa, 11.3 ml; CLC-ec 100 kDa, 12.5 ml; KcsA [17], 74 kDa, 13.3 ml.
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