The Enzymatic Iodination of the Red Cell Membrane

Abstract

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 . 10-157 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 106 iodide atoms per ghost does not alter this pattern . Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 4070 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 1251 and 131I does not reveal the appearance of previously unexposed proteins.

Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane

“…Only a small proportion of the total Vol. 161 available tyrosine sites on the cell surface are iodinated (Hubbard & Cohn, 1972), and thus the relative incorporation of the two iodine isotopes into each of the labelled extracellular peptides should reflect the relative accessibility of each tyrosine site to lactoperoxidase under iso-and hypo-osmotic conditions. The 'ghosts' were trypsin-treated under iso-osmotic conditions, and fragnents TI and T2 were isolated by polyacrylamide-gel electrophoresis (Fig.…”

Biosynthesis of Reticulocyte Membrane Proteins by Membrane-Free Polyribosomes

“…There exist 7-9 main components (1-8); the major species include two polypeptides of molecular weight greater than 200,000 (4), a protein of molecular weight 100,000, and a sialoylgylcoprotein which contains the MN, A, and B blood-group activities (8). The latter two species are the only membrane proteins found on the cell surface (3,(9)(10)(11)(12); they may also penetrate to the interior surface of the membrane (13)(14)(15). Little is known, however, about the biosynthesis of erythrocyte membrane proteins: the types of erythropoetic cells that synthesize each protein, the type of polysome (free or membrane-attached) that synthesizes the proteins, and the factors that regulate their synthesis.…”

Lactoperoxidase-catalysed iodination of surface proteins on human melanoma cells

“…It is intriguing that most of the other small proteins are resistant to trypsin. The possibility that these are intracellular proteins is considered unlikely, because the lactoperoxidase-catalysed iodination has now been well tested and shown to label only surface proteins (Hubbard and Cohn, 1972;1975). Furthermore, the autoradiographic patterns of the labelled cell-surface proteins bore no resemblance to the total cell protein pattern detected by Coomassieblue staining.…”

“…Melanoma cell lines HTC 163 and 312 have been described by Whitehead (1976 Jodintion. The method described by Hubbard and Cohn (1972) was used. Cell monolayers on plastic Petri dishes (5 cm diameter) were washed x3 with 4 ml phosphate buffered saline (PBS), pH 7 4, and then iodinated with a mixture containing 60 ,uCi of carrier-free 1251 (The Radiochemical Centre, Amersham, Bucks), 2-8 ,umol glucose, 5 u lactoperoxidase (Sigma Chemical Co., Kingstonupon-Thames, Surrey) and 3 u glucose oxidase (Sigma Chemical Co.) in 1 ml PBS for 15 min at room temperature.…”