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Solubilization and peptide "mapping" of a large external glycoprotein fraction labelled by lactoperoxidase-catalysed iodination of cultured fibroblasts.

A glycoprotein(s) of apparent molecular weight 220000-250000 can be labelled by lactoperoxidase-catalysed iodination of fibroblasts from a variety of species (Hynes, 1973 ; Hogg, 1974; Pearlstein & Waterfield, 1974; Yamada & Weston, 1974). The glycoprotein of hamster fibroblasts appears at the cell surface during interphase and is lost from the surface at mitosis (Hynes &Bye, 1974). Further, the glycoprotein is absent from the surface of virally transformed derivatives of hamster, chick, human and mouse fibroblasts (Hynes, 1974) and has been termed LETS (large external transformationsensitive) glycoprotein (Hynes & Bye, 1974). There are exceptions to this correlation, however (Meager et al., 1975). At present no function has been defined for the LETS glycoprotein, although its external location, apparently in a fraction related to the cell coat or glycocalyx (Graham et al., 1975), and relativity to growth state of the cells suggest a possible involvement in cellular processes mediated via the cell surface. To study further the structure and function of the LETS glycoprotein it is necessary to isolate the material in pure form. In this report we describe the solubilization characteristics of LETS glycoprotein and also show that extensive differences exist in the primary structures of the LETS glycoproteins obtained from chick or hamster fibroblasts. Baby-hamster kidney BHK21 C13 cells, hamster NIL 8 cells, primary chick-embryo fibroblasts and primary human skin fibroblasts were grown at 37°C in Glasgow-modified minimal essential medium supplemented with 10% (v/v) foetal bovine serum, 10% (v/v) tryptose phosphate broth,0.2 %(w/v) NaHC03, penicillin(0.1 M-i.u./l) andstreptomycin (0.1 g/l). Confluent monolayers of cells were labelled on 6Omm Falcon plastic culture dishes with a mixture of lactoperoxidase (Sigma Chemical Co., London S.W.6, U.K.) glucose oxidase (type V, Sigma) and carrier-free NalZSI (The Radiochemical Centre, Amersham, Bucks., U.K.) as described by Meager et al. (1975). After labelling, the cell sheets were washed with phosphate-buffered saline, pH7.4, scraped off with arubber policeman and finally dissolved in 1 %sodium dodecyl sulphate/ 1 % 2-mercaptoethanol by heating at 90°C for 5 min. Samples were analysed on sodium dodecyl sulphate/polyacrylamide gels by the discontinuous gel system described by Maize1 (1971). After resolution the gels were sliced into 2mm slices and counted directly for radioactive iodine associated with protein bands. As a routine the separations show a slow-moving major iodinated band (LETS glycoprotein) which is well separated from other faster-moving labelled bands. The LETS glycoprotein of BHK cells is not extracted into solution by either 0.5 % Nonidet (NP-40) (Fig. 1) or 1 % sodium deoxycholate. Extractions were carried out for up to 45min at 4°C. Notably, the other iodinated bands were readily solubilized under these conditions. These findings strongly suggest that the LETS glycoprotein is differently organized within the cell surface structure and is not released, as are the integral components, after extensive membrane disassembly with detergents. Similar behaviour has been described for non-integral membrane components such as spectrin (Steck, 1974). However, the LETS glycoprotein, unlike extrinsic components of the erythrocyte membrane, is also resistant to extraction with 0.2 % EDTA (results not shown). Under these conditions essentially no iodinated components are made soluble. The LETS