Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules.
A novel Mr 28,000 integral membrane protein ("28kDa") was identified in human erythrocytes and found entirely associated with the Triton X-100 insoluble membrane skeletons. Antibodies to 28kDa reacted strongly on immunoblots with 28kDa and a diffuse region of Mr 35,000-60,000 ("HMW-28kDa"). Selective proteolytic digestions of membranes demonstrated that HMW-28kDa has an extracellular domain, and both 28kDa and HMW-28kDa have intracellular domains. 28kDa and HMW-28kDa were purified to homogeneity. Quantitative immunoblots indicate that each erythrocyte contains 120,000-160,000 copies of 28kDa. Two-dimensional iodopeptide maps of 28kDa and HMW-28kDa were nearly identical; peptide-N-glycosidase digestion of purified HMW-28kDa demonstrated that it is the N-glycosylated form of 28kDa. When concentrated, 28kDa formed a series of larger oligomers which were stable in sodium dodecyl sulfate. Of several nonerythroid tissues studied with anti-28kDa immunoblots, only kidney displayed immunoreactive 28kDa. Purified rat kidney 28kDa was nearly identical to rat erythrocyte 28kDa when compared by two-dimensional iodopeptide mapping. Immunohistochemical staining of human kidney with anti-28kDa demonstrated prominent staining over the apical brush borders of proximal convoluted tubules. A novel integral membrane protein has been purified from erythrocyte and kidney membranes. This new protein may play a role in linkage of the membrane skeleton to the lipid bilayer.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D007163:Immunosorbent Techniques; D007684:Kidney Tubules / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats
D000596:Amino Acids; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol