The enzymatic iodination of the red cell membrane.
An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane. 97% of the incorporated isotope is in the erythrocyte ghost and 3% is associated with hemoglobin. No significant labeling of the red cell membrane occurs in the absence of LPO or by the deletion of any of the other reagents. A 6 million-fold excess of chloride ions inhibits iodination by no more than 50%. Incorporation of up to 1 x 10(6) iodide atoms into a single erythrocyte membrane results in no significant cell lysis. The incorporated label is exclusively in tyrosine residues as monoiodotyrosine. 10-15% of the trichloroacetic acid-precipitable radioactivity can be extracted with lipid solvents but is present as either labeled protein or (125)I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins reveals only two labeled protein bands out of the 15 present, and the presence of 50-1 x 10(6) iodide atoms per ghost does not alter this pattern. Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 40% of the total label. Component b has an apparent molecular weight of 74,000, contains all of the demonstrable sialic acid, and accounts for 60% of the total label. Trypsinization of iodinated, intact red cells results in the disappearance of only component b, the appearance of labeled glycopeptides in the medium, and the absence of smaller, labeled peptides remaining in the membrane. Pronase treatment hydrolyzes component b in a similar fashion, but also cleaves component a to a 72,000 mol wt peptide which is retained in the membrane. A combination of protease treatment and double labeling with (125)I and (131)I does not reveal the appearance of previously unexposed proteins.
D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism*; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide; D007454:Iodides / Q000378:metabolism*; D007456:Iodine Isotopes; D008722:Methods; D008970:Molecular Weight; D009439:Neuraminidase; D010447:Peptide Hydrolases; D010544:Peroxidases; D011402:Pronase; D014357:Trypsin; D014443:Tyrosine / Q000032:analysis
D007454:Iodides; D007456:Iodine Isotopes; D014443:Tyrosine; D006861:Hydrogen Peroxide; D010544:Peroxidases; D009439:Neuraminidase; D010447:Peptide Hydrolases; D014357:Trypsin; D011402:Pronase