Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies.
The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55,000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze-thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)
Burdett
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Department of Cell Biology, Hospital for Sick Children, Toronto, Ont., Canada.
Klip
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D000818:Animals; D000918:Antibody Specificity; D001786:Blood Glucose / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood*; D002460:Cell Line; D003571:Cytochalasin B / Q000378:metabolism; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D013552:Swine
D001786:Blood Glucose; D002352:Carrier Proteins; D003571:Cytochalasin B