ESTIMATE OF THE NUMBER OF UREA TRANSPORT SITES IN ERYTHROCYTE-GHOSTS USING A HYDROPHOBIC MERCURIAL

In this paper a variety of mercurials, including a pCMB-nitroxide anal ogue, were used to study urea transport in human red cell ghosts. It w as determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitrox ide, and chlormerodrin extended over four orders of magnitude consiste nt with their measured oil/water partition coefficients. From these re sults, we concluded that a significant hydrophobic barrier limits acce ss to the urea inhibition site, suggesting that the urea site is burie d in the bilayer or in a hydrophobic region of the transporter. In con trast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thu s, the water inhibition site may be more directly accessible via the a queous phase. Under conditions that leave water transport unaffected, we determined that less-than-or-equal-to 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 resu lt is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental numbe r of urea sites, a turnover number between 2-6 x 10(6) sec-1 at 22-deg rees-C is calculated suggesting a channel mechanism.