Kinetics of conformational changes associated with inhibitor binding to the purified band 3 transporter. Direct observation of allosteric subunit interactions.
Subunit interaction effects were identified for isolated human erythrocyte band 3, the anion exchanger, by observing both static and stopped-flow kinetic protein fluorescence changes associated with inhibitor binding to the intramonomeric stilbenedisulfonate site. We measured the rate of conformational changes associated with reversible binding of H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate). The rate of H2DIDS release was also measured. As a test for subunit interactions, we studied the effect of partial labeling of the band 3 monomer population with H2DIDS on the equilibrium and kinetics of H2DIDS reversible binding to the remaining monomers. The results showed biphasic kinetics for control band 3, with a pseudo-first-order ligand dependence for the fast phase followed by a slow ligand-independent relaxation. A second-order "on" rate constant for the fast phase was determined to be (1.2 +/- 0.1) x 10(7) M-1 s-1, while the associated "off" rate constant was found to be 1.1 +/- 0.5 s-1. From these kinetic constants, we calculated a Kd value of 95 +/- 50 nM, which is in excellent agreement with the Kd value determined at thermodynamic equilibrium (110 +/- 9 nM). Covalent labeling of 75% of the band 3 monomer population with H2DIDS changed the kinetics of the fast phase, slowing the apparent rate by changing the order of the reaction from pseudo-first-order to zero-order. Partial labeling did not affect the ligand-independent relaxation. Separate measurements of the H2DIDS "off" rate also showed a biphasic time course, with a 20-fold difference in apparent rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)
Salhany
J M
JM
Veterans Administration Medical Center, Omaha, Nebraska.
Cordes
K A
KA
Schopfer
L M
LM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D000495:Allosteric Site; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001667:Binding, Competitive; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics
D001457:Anion Exchange Protein 1, Erythrocyte; D046911:Macromolecular Substances; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid