Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial

Abstract

In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that < or = 32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2-6 x 10(6) sec-1 at 22 degrees C is calculated suggesting a channel mechanism.

The permeability of red blood cells to chloride, urea, and water

“…The affinity to urea in these cells is even higher than in dog RBC as K ½ is 127mmoll ) becomes similar: 1.6, 0.9 and 1.2 in Amphiuma, dog and human RBC, respectively. In human RBC UT-B is ascribed to be the Kidd antigen and the estimated number of transporters is between 14,000 and 32,000 (Masouredis et al, 1980;Fröhlich et al, 1991;Mannuzzu et al, 1993;Neau et al, 1993;Olivès et al, 1995). Taking the 14,000 copies, the turnover number in human RBC is 5×10 6 ureamoleculessite -1 s -1 at 25°C, which is in agreement with previous estimates (Mannuzzu et al, 1993;Sands et al, 1997).…”

“…In human RBC UT-B is ascribed to be the Kidd antigen and the estimated number of transporters is between 14,000 and 32,000 (Masouredis et al, 1980;Fröhlich et al, 1991;Mannuzzu et al, 1993;Neau et al, 1993;Olivès et al, 1995). Taking the 14,000 copies, the turnover number in human RBC is 5×10 6 ureamoleculessite -1 s -1 at 25°C, which is in agreement with previous estimates (Mannuzzu et al, 1993;Sands et al, 1997). It is an open question whether the similar J urea max,cell in the three species is due to the same number of transport sites per cell with the same turnover rate per site or a different number of transport sites with different turnover rates per site.…”

“…It is an open question whether the similar J urea max,cell in the three species is due to the same number of transport sites per cell with the same turnover rate per site or a different number of transport sites with different turnover rates per site. The turnover number indicates a channel-like mechanism (Mannuzzu et al, 1993), but the term 'facilitated diffusion' conveniently reflects the effect and not the mechanism of UT-B.…”

Functional characterization ofActinobacillus pleuropneumoniaeurea transport protein, ApUT

“…The measured turnover numbers for UT-B has been reported to be two to three orders of magnitude faster than the fastest known carrier (erythrocyte anion exchanger). Accordingly, a channel-mediated mechanism was suggested for UT-B mediated urea transport (19). The apparent saturability of ApUT indicates that rather than acting as a simple pore, urea is transported by this transporter either by a carrier-mediated mechanism or by a simple channel process involving a binding site for urea.…”

“…The apparent saturability of ApUT indicates that rather than acting as a simple pore, urea is transported by this transporter either by a carrier-mediated mechanism or by a simple channel process involving a binding site for urea. To distinguish between a channel or a simple carrier mode of transport, additional specific measurements of single-channel fluxes or turnover numbers are required (19,36). The measured single-channel fluxes of mammalian urea transporters UT-A2 and UT-A3 are 100-fold less than UT-B, the erythrocyte urea transporter, and did not show apparent saturation kinetics, which suggest a channel mode of urea transport for these transporters (18).…”