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 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.
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
L M
LM
Mannuzzu
M M
MM
Moronne
R I
RI
Macey
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002717:Chlormerodrin / Q000494:pharmacology; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D013113:Spin Labels; D014508:Urea / Q000097:blood*; D014867:Water / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D013113:Spin Labels; D014867:Water; D020245:p-Chloromercuribenzoic Acid; D002731:4-Chloromercuribenzenesulfonate; D014508:Urea; D002717:Chlormerodrin