Functional roles of carboxyl groups in human red blood cell anion exchange.
Chemical modification and proteolytic digestion have been used to examine three aspects of the role of carboxyl groups in the anion transport catalyzed by the red cell inorganic anion-exchange protein (known as band 3 or capnophorin). The modification employs the negatively charged Woodward's reagent K and BH4, which together can convert protein carboxyl groups to alcohols (Jennings and Anderson, 1987). The reagent, with radioactive BH4, can be used to label reactive carboxyl groups in intact cells under conditions in which only extracellular residues are labeled. Carboxyl groups in both major chymotryptic fragments are labeled, with most of the label in the 35-kD COOH-terminal fragment. There is significant labeling in the 28-kD papain fragment, but not in the 7-kD papain fragment. Papain itself does not inhibit the proton-SO4 cotransport that takes place during Cl/SO4 exchange. These results all indicate that the carboxyl group associated with proton-SO4 cotransport is not removed by papain and is in the COOH-terminal 28-kD fragment. A set of experiments was performed to determine the effect of the Cl gradient on the labeling of both the 60-kD and 35-kD chymotryptic fragments. This result provides evidence against the idea that either of the reactive carboxyl groups participates in a "zipper" transport mechanism, in which anions and protein-bound negative charge move in opposite directions. Finally, conversion of band 3 carboxyl groups to alcohols (again under conditions of minimal permeation of the reagent) causes a nearly 10-fold increase in stilbene disulfonate-sensitive Cl conductance. This indicates that a reactive carboxyl group constitutes part of the permeability barrier to net conductive anion transport through band 3. This carboxyl group may be the same as the one associated with proton-anion cotransport.
Jennings
M L
ML
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston.
Anderson
M P
MP
McCormick
S J
SJ
D000837:Anion Exchange Resins / Q000378:metabolism; D000838:Anions / Q000378:metabolism*; D002264:Carboxylic Acids / Q000032:analysis / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism* / Q000502:physiology; D006801:Humans
D000837:Anion Exchange Resins; D000838:Anions; D002264:Carboxylic Acids