Structural and functional characterization of band 3 from Southeast Asian ovalocytes.
To determine why deletion of the nine amino acids joining the membrane and cytoplasmic domains of band 3 from Southeast Asian ovalocytes (SAO) renders the erythrocytes rigid, we compared the structural and functional properties of SAO and normal band 3. Calorimetric data, inhibitor binding studies, and anion transport assays all reveal that the membrane-spanning domain of SAO band 3 is denatured, while proteolysis studies and circular dichroism spectroscopy suggest the mutant domain retains much secondary structure. It is concluded that the transmembrane helices of SAO band 3 are dissociated and randomized but not unfolded. The cytoplasmic domain of SAO band 3 was shown to be structurally and functionally normal based on (i) calorimetric properties, (ii) native conformational change, (iii) ability to form an intersubunit disulfide bond, (iv) affinity and capacity for binding ankyrin and protein 4.1, and (v) kinetics of association with ankyrin. However, both normal and mutant isoforms of band 3 in SAO cells were found to adhere nonspecifically to the spectrin skeleton. Further, when SAO cells were osmotically swollen, the detergent extractability of band 3 became normal. We propose that much of band 3 is nonspecifically entrapped in the spectrin network in SAO cells and that this nonspecific adhesion may be responsible for the rigidity of the SAO erythrocyte.
Moriyama
R
R
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Ideguchi
H
H
Lombardo
C R
CR
Van Dort
H M
HM
Low
P S
PS
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017487:Ankyrins / Q000378:metabolism; D001210:Asia, Southeastern; D001483:Base Sequence; D002151:Calorimetry; D002942:Circular Dichroism; D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D004220:Disulfides / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000502:physiology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012016:Reference Values; D017384:Sequence Deletion
D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D004220:Disulfides; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D004247:DNA