Submitted on August 26, 2008
Revised on January 8, 2009
Accepted on January 8, 2009
Functional Characterization of Actinobacillus pleuropneumoniae Urea Transport Protein, ApUT
Geeta Godara1, Craig P. Smith2, Janine Bosse3, Mark L. Zeidel4, and John C Mathai4*
1 Maine Medical Center
2 University of Manchester
3 Imperial college
4 Beth Israel Deaconess Medical Center
* To whom correspondence should be addressed. E-mail: jmathai{at}bidmc.harvard.edu.
Urea transporters (UTs) effect rapid flux of urea across biological membranes. In the mammalian kidney, UT activity is essential for effective urine concentration. In bacteria, UT-mediated urea uptake permits intracellular urease to degrade urea to ammonia and CO2, a process which either buffers acid loads or provides nutrient nitrogen. We have characterized the urea transport channel protein ApUT from Actinobacillus pleuropneumoniae. Kinetic analysis of bacterial inside-out membranes enriched in ApUT showed ~28 fold increase in urea permeability (3.3 ± 0.4 x10-4 cm/s) compared to control vesicles (0.11 ± 0.02 x10-4 cm/s). In addition to urea ApUT also conducts water. Urea and water transport across the channel was phloretin and mercury inhibitable and the site of inhibition may be located on the cytoplasmic side of the protein. Glycerol and urea analogues such as methylamine, dimethylurea, formamide, acetamide, methylurea, propanamide and ethylamine did not permeate across ApUT.
