Alterations in pyridoxal 5'-phosphate inhibition of human erythrocyte anion transport associated with osmotic hemolysis and resealing.
When pyridoxal 5'-phosphate (PLP) is covalently bound to band 3 protein in intact red blood cells and those cells are subjected to the osmotic hemolysis and resealing process, a significant reduction in the original PLP anion transport inhibitory potency occurs. We show that partial deinhibition is not due to the development of a second anion transport pathway in resealed ghosts. Rather, partial deinhibition arises from a hemolysis-induced conformational change in CH17 (17-kDa integral chymotryptic domain of band 3). This change causes the extracellular exposure of new transport inhibitory sites. Exposure of the new sites leads to a 2-fold increase in PLP labeling of CH17 in resealed ghosts compared with CH17 in intact red cells. The hemolysis and resealing process has no effect on the labeling of CH35 (35-kDa integral chymotryptic fragment of band 3). Double-labeling studies show restoration of transport inhibitory potency to near red cell levels when the newly exposed CH17 sites are labeled with PLP in resealed ghosts. The results support the view that CH17 contains PLP transport inhibitory sites. They show that a major conformational change occurs in band 3 with hemolysis.
Salhany
J M
JM
Veterans Administration Medical Center, Omaha, Nebraska 68105.
Rauenbuehler
P B
PB
Sloan
R L
RL
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D000838:Anions; D001692:Biological Transport / Q000187:drug effects; D004227:Dithionite / Q000097:blood; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000494:pharmacology*
D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D004227:Dithionite; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D011732:Pyridoxal Phosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid