Associations between erythrocyte band 3 protein and aldolase in detergent solution. Determining their stoichiometry by analytical ultracentrifugation.
The cytoplasmic domain of band 3, the predominant polypeptide of the erythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human band 3 protein and aldolase, in order to clarify the role of the different band 3 oligomers as ligand binding sites. The experiments were performed on mixtures of solubilized band 3 and aldolase in solutions of a nonionic detergent, nonaethyleneglycol lauryl ether. The main technique applied was sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To facilitate the evaluations, the aldolase was labelled with a dye. The following results were obtained. (1) With unmodified band 3, aldolase is bound exclusively or at least predominantly to the band 3 tetramer (but not to monomers or dimers). (2) The band 3 tetramer can bind up to four aldolase tetramers. (3) The band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable band 3 dimers (stabilized either covalently or noncovalently) can also associate with aldolase and can bind up to two aldolase tetramers. The results described, together with those reported previously, point at a prominent role of the band 3 tetramer in ligand binding.
Huber
E
E
Institut für Biophysik, J. W. Goethe-Universität, Frankfurt am Main, Germany.
Bäumert
H G
HG
Spatz-Kümbel
G
G
Schubert
D
D
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D003902:Detergents; D019281:Dimerization; D004910:Erythrocyte Membrane; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D011485:Protein Binding; D011817:Rabbits; D012996:Solutions; D014461:Ultracentrifugation
D001457:Anion Exchange Protein 1, Erythrocyte; D003902:Detergents; D012996:Solutions; D005634:Fructose-Bisphosphate Aldolase