Abstract
In an attempt to identify potential regulatory mechanisms for erythrocyte membrane-cytoskeletal interactions, the kinetics and pH dependence of the band 3-ankyrin interaction were investigated. Association of 125I-ankyrin with KI-stripped inside-out erythrocyte membrane vesicles was found to proceed in two kinetic phases. The initial, fast phase (t1/2 approximately 15-30 min) involved predominantly the binding of ankyrin to low affinity sites (KD approximately 130 nM) in a pH-dependent manner. The apparent pKa values describing this reversible pH dependence (7.2 +/- 0.1 and 9.2 +/- 0.1) defined states of band 3 with high, moderate, and no capacity to bind ankyrin (in order of increasing pH). Since the cytoplasmic domain of band 3 also exists in 3 distinct conformational states characterized by apparent pKa values of 7.2 and 9.2, it was hypothesized that the reversible structural equilibrium in band 3 could influence ankyrin binding. The second or slow phase of ankyrin binding to band 3 involved the conversion of low to high affinity sites (KD approximately 13 nM). This phase, which was largely temperature and pH independent, required roughly an order of magnitude longer to reach completion than the fast phase. Unfortunately, even though the slow phase could be cleanly separated from the fast phase at low pH, insufficient data were available to formulate a physical interpretation of its origin. Significantly, however, even after completion of the slow phase under the most quantitative binding conditions identified, a maximum of only 26% of the band 3 was found to bind ankyrin in situ. Although higher ankyrin-band 3 stoichiometries may be achievable with the isolated cytoplasmic fragment of band 3, we interpret the above 1:4 stoichiometry to suggest that the tetramer of band 3 constitutes the predominant ankyrin binding oligomer of band 3 on the membrane.
Highlights
We have shown that binding of ankyrin to stripped insideout erythrocyte membrane vesicles occurs in two kinetic phases
As pH was raised from pH 6 to 10, the membrane capacity for ankyrin decreased from a high level to a moderate level and to a negligible level in three continuous steps characterized by apparent pK, values of 7.2 and 9.2
We have hypothesized that the pHdependent conformational equilibrium in the cytoplasmic domain of band 3, which is triphasic and characterized by apparent pKGl values of 7.2 and 9.2, is responsible for this binding sensitivity to pH
Summary
Extracted inside-out membrane vesicles (KI-IOV) were prepared essentially as described by Bennett [33]. Prior to storage the washed KI-IOV were suspended to about 2 mg/ml in the above buffer, centrifuged for 10 min at 40,000 x g, and separated from both a hard darker pellet and a floating cloudy fraction. Ankyrin (-2 mg in 1 ml) was typically radiolabeled to a specific activity of about 100,000 cpm/Fg with 1 mCi of ‘251-labeled Bolton-Hunter reagent, as described [33]. The stock of “‘I-ankyrin was generally centrifuged for 2 h at 47,000 x g to remove any pelletable material before use
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
