Abstract
Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.
Highlights
Of the amino terminal and 480-492 of the carboxyl and carboxyl (CT) terminals [2]
The peptides were conjugated to keyhole limpet hemocyanin, and each of the conjugates was used to immunize three rabbits [9].The sera were tested for reactivity with unconjugated homologous and heterologous peptides, and purified human erythrocyte transporter [10] by solid-phase radioimmunoassay [11].All studies reported utilized antiserum elicited to carboxyl terminaldomain of the proteinmay beuseful either pCT (a-CT) or pNT (a-nearly identical amino (NT)) from the second bleeds of the for theimmunocytochemicallocalization of the glucose same immunized rabbit for each respective peptide except for imtransporter
Two of the sera from rabbits immunized with pCT and none of the sera from the rabbits immunized with pNT reacted with purified human erythrocyte glucose transporter
Summary
Of the amino terminal and 480-492 of the carboxyl and carboxyl (CT) terminals [2]. Hydropathy plots predict terminal of the deduced sequence of the glucose trans- that theglucose transporter proteinmay have 12membraneporter were synthesized and used to produce site-spe- spanning regions and that its NT and CThave cytoplasmic cific polyclonal antipeptidesera. The antiserum to the carboxyl terminal immunoblots the M , 55,000 glucose transporter in erythrocyte membranes andthepurified erythrocytetransporter.
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