Rotational diffusion of the erythrocyte integral membrane protein band 3: effect of hemichrome binding.
Human erythrocyte band 3 was covalently labeled within the integral membrane domain by incubating intact erythrocytes with the phosphorescent probe eosinyl-5-maleimide. The rotational diffusion of band 3 in membranes prepared from these labeled cells was measured using the technique of time-resolved phosphorescence anisotropy. Three rotational correlation times ranging from 16 to 3800 microseconds were observed, suggesting that band 3 exists in different aggregate states within the plane of the membrane. The oxidizing agent phenylhydrazine was used to induce hemichrome formation within intact erythrocytes. The immobilization of band 3 in membranes prepared from these erythrocytes suggests that the binding of hemichromes induces clustering of band 3. The addition of purified hemichromes to erythrocyte ghosts leads to a similar effect. We have also examined the mobility of the cytoplasmic domain of band 3. This region was labeled indirectly using a phosphorescently labeled antibody which binds to an epitope within the cytoplasmic domain. We observed very rapid motion of the cytoplasmic region of band 3, which was only partially restricted upon hemichrome binding. This suggests that the integral and cytoplasmic domains of band 3 may be independently mobile.
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
R A
RA
McPherson
W H
WH
Sawyer
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L
Tilley
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry; D003599:Cytoskeleton / Q000737:chemistry; D004058:Diffusion; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000187:drug effects; D005454:Fluorescence Polarization; D006420:Hemeproteins / Q000737:chemistry*; D006801:Humans; D010659:Phenylhydrazines; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D001457:Anion Exchange Protein 1, Erythrocyte; D006420:Hemeproteins; D010659:Phenylhydrazines; C001332:hemichrome; C030299:phenylhydrazine; C033531:eosin maleimide; D004801:Eosine Yellowish-(YS)