Functional carboxyl groups in the red cell anion exchange protein. Modification with an impermeant carbodiimide.
Anion exchange in human red blood cell membranes was inactivated using the impermeant carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide (EAC). The inactivation time course was biphasic: at 30 mM EAC, approximately 50% of the exchange capacity was inactivated within approximately 15 min; this was followed by a phase in which irreversible exchange inactivation was approximately 100-fold slower. The rate and extent of inactivation was enhanced in the presence of the nucleophile tyrosine ethyl ester (TEE), suggesting that the inactivation is the result of carboxyl group modification. Inactivation (to a maximum of 10% residual exchange activity) was also enhanced by the reversible inhibitor of anion exchange 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) at concentrations that were 10(3)-10(4) times higher than those necessary for inhibition of anion exchange. The extracellular binding site for stilbenedisulfonates is essentially intact after carbodiimide modification: the irreversible inhibitor of anion exchange 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) eliminated (most of) the residual exchange activity: DNDS inhibited the residual (DIDS-sensitive) Cl- at concentrations similar to those that inhibit Cl- exchange of unmodified membranes: and Cl- efflux is activated by extracellular Cl-, with half-maximal activation at approximately 3 mM Cl-, which is similar to the value for unmodified membranes. But the residual anion exchange function after maximum inactivation is insensitive to changes of extra- and intracellular pH between pH 5 and 7. The titratable group with a pKa of approximately 5.4, which must be deprotonated for normal function of the native anion exchanger, thus appears to be lost after EAC modification.
Bjerrum
P J
PJ
Department of General Physiology and Biophysics, University of Copenhagen, Panum Institute, Denmark.
Andersen
O S
OS
Borders
C L
CL
Wieth
J O
JO
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D027321:Anion Transport Proteins; D000838:Anions / Q000378:metabolism / Q000494:pharmacology; D002234:Carbodiimides / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D004347:Drug Interactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010539:Permeability; D010658:Phenylglyoxal / Q000494:pharmacology
D027321:Anion Transport Proteins; D000838:Anions; D002234:Carbodiimides; D002352:Carrier Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D010658:Phenylglyoxal; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid