Gel filtration chromatographic studies of the isolated membrane domain of band 3.
We have investigated the oligomeric state of the membrane domain of band 3 (MDB3) in non-ionic detergent solution using Sepharose CL-4B gel filtration chromatography to study the hydrodynamic properties of the protein as a function of its concentration. The studies were performed in a C12E9 (polyoxyethylene-9-lauryl ether) buffer containing phosphatidylcholine and sodium chloride, which significantly slow a dilution-induced band 3 conformational change, and an associated aggregation process. Under these conditions native MDB3 eluted predominantly as a single Gaussian peak with a Stokes radius of 76 +/- 14 A, at all protein concentrations studies between 0.2 and 12 microM. This value agrees with the calculated Stokes radius (74 A) determined from the crystal structure of the MDB3 dimer. The Stokes radius of the MDB3 monomer was obtained experimentally by treating native MDB3 with 0.5% SDS, and exchanging the SDS for C12E9 on the Sepharose column. SDS-treated MDB3 showed two peaks whose ratio was strongly dependent on applied protein concentration. The peak representing the largest material had a Stokes radius of 69.7 +/- 14 A, which is essentially the same as the native MDB3 dimer. The peak representing the smaller material had a Stokes radius of 36 +/- 9 A, and was assigned as the MDB3 monomer in C12E9. Evidence is discussed which indicates that the C12E9 monomer specifically self-associates to form a functional MDB3 dimer. We conclude that native MDB3 exists as a stable dimer in mixed micellar solutions composed of C12E9 and phosphatidylcholine, and that the dimer can be dissociated to monomers only by denaturation.
Salhany
J M
JM
Veterans Administration Medical Center Omaha, Nebraska, USA.
Cordes
K A
KA
Sloan
R L
RL
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000737:chemistry / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism*; D002021:Buffers; D002850:Chromatography, Gel / Q000379:methods; D019281:Dimerization; D004801:Eosine Yellowish-(YS) / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D001457:Anion Exchange Protein 1, Erythrocyte; D002021:Buffers; D000077423:Polidocanol; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D004801:Eosine Yellowish-(YS)