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EXTERNALLY DISPOSED PLASMA MEMBRANE PROTEINS I. Enzymatic Iodination of Mouse

By L Cells, Ann L. Hubbard and Zanvil A. Cohn

Abstract

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. lodination 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 125I, 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. The introduction of reagents which specifically react with externally disposed constituents of the plasma membrane provides a method for probing an interesting domain of the cell's surface and for following membrane flow and degradation (2, 5, 8

Year: 1975
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