Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells.

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.

Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells.

“…Use of the enzymatic iodination technique to label only the externally disposed membrane proteins provided a reasonable solution to both problems. In the preceding report (16), we demonstrated that monoiodotyrosine (MIT)J a nonreutilisable amino acid (27), represents at least 90% of the iodinated species when intact L cells are labeled with the lactoperoxidase (LPO) system. In addition, 90% of the label has been localized to the plasma membrane after iodination, so any decrease in cell-associated radioactivity with time necessarily represents loss of label from those proteins originally present in the plasma membrane compartment.…”

Genetics of the large, external, transformation-sensitive (LETS) protein: assignment of a gene coding for expression of LETS to human chromosome 8.

“…1). Lactoperoxidase, a macromolecule of about 150,000 molecular weight, does not cross the lipid bilayer of intact cells, and therefore under appropriate conditions can catalyze the iodination of only externally disposed cell surface proteins (27,28). Individual proteins were classified by molecular weight as determined with molecular weight standards by NaDodSO4/polyacrylamide gel electrophoresis.…”

Surface proteins in normal and transformed rat liver epithelial cells in culture

“…Surface labelling.-Subconfluent cultures were surface iodinated according to the method of Hubbard & Cohn (1975). The reaction mixture contained 38 ,tg/ml lactoperoxidase (Sigma, St. Louis, Mo., U.S.A.) 0-6 u/ml glucose oxidase (Sigma type VII) 2-5 mg/ml glucose and 0-125 mCi/ml Na125I carrier-free (Amersham, U.K.) in phosphatebuffered saline (or in Williams's E medium plus FCS).…”