Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells.
The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.
D000818:Animals; D001345:Autoradiography; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005949:Glucose Oxidase; D007454:Iodides / Q000378:metabolism; D007457:Iodine Radioisotopes; D007739:L Cells / Q000032:analysis* / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000032:analysis; D010455:Peptides / Q000032:analysis; D010544:Peroxidases; D010727:Phosphoric Diester Hydrolases / Q000032:analysis; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification; D013347:Subcellular Fractions / Q000032:analysis; D013429:Sulfatases / Q000032:analysis
D007454:Iodides; D007457:Iodine Radioisotopes; D009363:Neoplasm Proteins; D010455:Peptides; D011506:Proteins; D002784:Cholesterol; D005949:Glucose Oxidase; D010544:Peroxidases; D010727:Phosphoric Diester Hydrolases; D013429:Sulfatases