Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes.
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.
Karniski
L P
LP
Department of Internal Medicine, University of Iowa, Iowa City.
Jennings
M L
ML
D000199:Actins / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal; D002417:Cattle; D003850:Deoxyribonuclease I; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D004912:Erythrocytes / Q000032:analysis; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007114:Immunization; D007668:Kidney / Q000032:analysis* / Q000648:ultrastructure; D037102:Lectins / Q000378:metabolism; D008297:Male; D008871:Microvilli / Q000032:analysis; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits
D000199:Actins; D000596:Amino Acids; D001457:Anion Exchange Protein 1, Erythrocyte; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007106:Immune Sera; D037102:Lectins; D011480:Protease Inhibitors; D003850:Deoxyribonuclease I; D004229:Dithiothreitol