Characterization of pyridoxal 5'-phosphate affinity labeling of band 3 protein. Evidence for allosterically interacting transport inhibitory subdomains.
Pyridoxal 5'-phosphate (PLP) is a substrate of band 3, the erythrocyte anion transport protein. It competitively inhibits anion transport and labels two exofacial chymotryptic domains (the 17-kDa (CH17) and the 35-kDa (CH35) integral fragments). Two mol of PLP are bound/mol of each fragment at saturation. PLP labeling of both domains is competitive with chloride at constant ionic strength. Addition of DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), protects PLP labeling of CH35 but exposes new, nonoverlapping sites on CH17.4,4'-Diisothiocyanostilbene-2,2'-disulfonate reduces PLP labeling to both domains with time, while NAP-taurine (N(-4-azido-2-nitrophenyl)2-aminosulfonate) has no effect on either domain. At low chloride (balance citrate) and high DNDS, we can strongly suppress CH35 labeling and selectively titrate CH17 with PLP. Correlation of fractional transport inhibition with fractional PLP covalent coverage of CH17, quantitatively follows the 1:2 correlation line indicating that full coverage of CH17 sites (which constitute half of the total PLP-labeling sites on band 3) exactly inhibits one-half of transport. PLP labeling of CH35 sites accounts for the other half of inhibition. The inhibition-labeling correlation plots are nonlinear in the absence of DNDS, indicating the presence of allosteric interactions between the domains. We conclude that CH17 and CH35 compose nonoverlapping, functionally equivalent, allosterically linked transport inhibitory subdomains on band 3.
Salhany
J M
JM
Veterans Administration Medical Center, Omaha, Nebraska 68105.
Rauenbuehler
P B
PB
Sloan
R L
RL
D000345:Affinity Labels; D000494:Allosteric Regulation; D000495:Allosteric Site; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001665:Binding Sites; D002712:Chlorides / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D011732:Pyridoxal Phosphate