A Comparison of Membrane Proteins of Normal and Transformed Cells by Lactoperoxidase Labeling

Abstract

The enzyme lactoperoxidase (iodide: hydrogen-peroxide oxidoreductase, EC 1.11.1.8) was used to iodinate ('2I) accessible proteins on membranes of intact virally transformed and untransformed cells. A number of labeled bands of proteins were detected by acrylamide gel electrophoresis. A heavily labeled band with a molecular weight of approximately 250,000 daltons was found in all untransformed cells but was absent from transformed cells. When Coomassie-blue-stained membrane preparations were compared, a band was seen in normal cells which comigrated with the lactoperoxidase-labeled band. In transformed cell membranes, three discrete bands were present in the same position. Thus, the expression of this protein may be altered when cells are in the transformed state.The identification of alterations in membrane proteins and glycoproteins after transformation by DNA or RNA oncogenic viruses has been confusing. In general, there is reported to be an overall decrease in numbers of membrane glycoproteins (1, 2), but there are demonstrations of an increased glycosylation of a restricted number of membrane glycoproteins in transformed cells (3, 4). Conversely, other studies have suggested an association with untransformed cell membranes (5), or their growth media (2), of glycoproteins which were absent from membranes of transformed cells.It was considered that a fruitful approach to resolving some of these complexities would be to examine directly the membranes of intact cells rather than isolated membrane fragments. The lactoperoxidase (iodide: hydrogen-peroxide oxidoreductase, EC 1.11.1.8) catalyzed iodination procedure (6) is a technique by which tyrosine-containing proteins exposed on the surface of cell membranes can be directly labeled. This technique has been used to examine proteins on the membranes of erythrocytes (6, 7), lymphocytes (8, 9), platelets (10, 11), fibroblasts (12), and viruses (13,14).In this study, lactoperoxidase-catalyzed iodination has been used to characterize the membranes of intact normal cell lines and their transformed derivatives. It was hoped that the iodination procedure was sufficiently mild that membrane architecture remained relatively unperturbed. MATERIALS AND METHODSCells. The cell lines used were BALB/C 3T3 (clone A31), Swiss 3T6, and secondary cultures of mouse-embryo fibroblasts from Taylors Own mice (ICRF), referred to as TO cells. All transformed cells used were derivatives of the 3T3 line A31: Py-3T3 and SV-3T3 were isolated after infection of 3T3 cells by the DNA viruses, polyoma and SV-40 viruses, respectively, and were a gift from Dr. Mike Fried. Transformed cells MSV-3T3 were isolated from a single infection of 3T3 by murine leukemia virus (Moloney pseudotype) which contained both sarcoma and leukemia virus activity. MSV-3T3 cells had a transformed morphology and were releasing sarcoma virus, as detected by the focus-forming assay, and leukemia virus, as detected by the XC plaque assay (15). The LX cells were a kind gift from Dr. Mike Shodell.All the cell lines, e...

Absence of fibronectin and presence of plasminogen activator in both normal and malignant human mammary epithelial cells in culture.

“…16). These data agree well with previous reports on surface expression of Fibronectin by these cells (12,16). total of I 1 primary cultures derived from 6 donors were tested .…”

A polyoma mutant that encodes small T antigen but not middle T antigen demonstrates uncoupling of cell surface and cytoskeletal changes associated with cell transformation.

“…Its disappearance associated with transformation has been studied extensively (16,35). Mouse 3T3 cells transformed by polyoma or simian virus 40 show reduced quantities of this protein (32). We close proximity to actin cables and adhesion sites (8,17,37,65).…”

Surface Labelling of Senescent Chick Fibroblasts by Lactoperoxidase-Catalysed Iodination

“…Lactoperoxidase catalysed iodination of intact cells has been shown by several groups to be relatively specific for labelling of exposed surface po lypeptides [4][5][6], Cytoplasmic proteins are not labelled to any significant extent [6]. In general several discrete iodinated components are recog nized after labelling of various cell types including hamster and mouse es tablished fibroblast lines [4,5], and human tumour KB cells [6].…”