Abstract
A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.
Highlights
A monoclonal antibody against the membrane do- ion exchange can proceed in theabsence of the NHp-terminal main of human erythrocyte band 3 was tested for its cytoplasmic domain [6]
A single brush border protein with a molecular mass tified in a number of non-erythroid tissues
It has been proposed that these cells take part in acidwith purified band 3 from human, rabbit, andbovine erythrocytes confirming the immunologic similarity among these proteins
Summary
Materials-Affi-Gel 10 was purchased from Bio-Rad, octyl glucoside from Behring Diagnostics, and prestained proteinstandards from Bethesda Research Laboratories. After centrifugation a t 500 X g for 30 s, the supernatant was removed from the beads and diluted 30-fold into 100 mM sucrose, 5 mM HEPES/Tris,pH 7.5 (SHT buffer) and spun a t 15,000 rpm for 25 min in a Sorval SA 600 rotor For those experiments where solubilized DNase I membranes were used, the pellet containing the DNase I-purified brush border membrane fraction was resuspended in SHT buffer and mixed with an equal volume of 4% octyl glucoside in SHT on ice for 2 h. Lectin Blots-The nitrocellulose membranes were blocked with PBS-Tween for 1h a t room temperature, incubated witheither peroxidase-conjugated wheat germ agglutinin or peroxidase-conjugated concanavalin A.(1:10,000 dilution) for 2 h. The membranes were washed twice in PBS-Tween, rinsed several times in distilled water, developed with the same solution described above for immunoblots. Amino acid determination was performed on aBeckman model 7300 amino acid analyzer at theYale University Protein and Nucleic Acid Chemistry Facility
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