Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, band 3.
Solution studies of the cytoplasmic domain (molecular mass approximately 40kDa) of band 3, the anion exchanger from human erythrocyte membranes, previously suggested a dimeric molecule on the basis of the relative techniques of calibrated gel filtration and calibrated preparative ultracentrifugation. This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography and absolute ultracentrifugation techniques. Sedimentation velocity in the analytical ultracentrifuge combined with calibrated gel chromatography give a molecular mass M of (77 +/- 4) kDa, a value confirmed by low-speed sedimentation equilibrium. Velocity sedimentation in the analytical ultracentrifuge gave a single sedimenting species with an s o 20,w of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation constant Kd, with a value from direct fitting of the concentration distribution curves of (2.8 +/- 0.5) microM, confirmed by a value of approximately 3 microM obtained from fitting a plot of molecular weight Mw,app versus cell loading concentration. Hydrodynamic calculations based on the classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of approximately 10:1, consistent with observations from electron microscopy.
Cölfen
H
H
National Centre for Macromolecular Hydrodynamics, University of Nottingham, Sutton Bonington, UK.
Harding
S E
SE
Boulter
J M
JM
Watts
A
A
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D055592:Biophysical Phenomena; D001703:Biophysics; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000737:chemistry; D019281:Dimerization; D004910:Erythrocyte Membrane / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D015394:Molecular Structure; D008970:Molecular Weight; D011487:Protein Conformation; D012996:Solutions
D001457:Anion Exchange Protein 1, Erythrocyte; D012996:Solutions