Abstract
We have previously shown that lactoperoxidase (LPO) covalently coupled to polystyrene tissue culture flasks can be used to radioiodinate monolayer cell proteins that come into intimate contact with the LPO- polystyrene surface. These studies have now been extended to include a detailed examination of the class of iodinated polypeptides migrating with apparent molecular weights of 50,000 and 55,000 in SDS polyacrylamide gels. Whereas in cultured L929 cells the 55,000 band is predominantly iodinated, in thioglycollate-activated murine peritoneal macrophages the 55,000 and 50,000 bands are of equal intensity. It is possible that the marked degree of exposure of the 50,000 mol wt polypeptide to immobilized LPO is related to the unique strength of macrophages attachment. After labeling of both L929 cells and macrophages with immobilized LPO, all polypeptides in this molecular weight region were subjected to peptide mapping by simultaneous limited proteolysis and electrophoresis in a second SDS polyacrylamide slab gel. The results clearly show that the two major polypeptides in this region are identical within the limits of resolution of this technique. The 55,000 mol wt polypeptide can also be identified in Triton X-100 cytoskeletons from L929 cells after labeling with soluble LPO either before or after detergent lysis. We conclude that this cell surface polypeptide is in continuity with the cytoskeleton and is preferentially exposed to the substratum during attachment to polystyrene.
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Selected References
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- Ben-Ze'ev A., Duerr A., Solomon F., Penman S. The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins. Cell. 1979 Aug;17(4):859–865. doi: 10.1016/0092-8674(79)90326-x. [DOI] [PubMed] [Google Scholar]
- Brown S., Levinson W., Spudich J. A. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct. 1976;5(2):119–130. doi: 10.1002/jss.400050203. [DOI] [PubMed] [Google Scholar]
- Chin N. W., Lanks K. W. Covalent attachment of lactoperoxidase to polystyrene tissue culture flasks. Anal Biochem. 1977 Dec;83(2):709–719. doi: 10.1016/0003-2697(77)90076-8. [DOI] [PubMed] [Google Scholar]
- Chin N. W., Lanks K. W. Use of immobilized lactoperoxidase to label L cell proteins involved in adhesion to polystyrene. J Cell Biol. 1980 May;85(2):402–413. doi: 10.1083/jcb.85.2.402. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
- Evans R. M., Ward D. C., Fink L. M. Asymmetric distribution of plasma membrane proteins in mouse L-929 cells. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6235–6239. doi: 10.1073/pnas.76.12.6235. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geiger B., Singer S. J. The participation of alpha-actinin in the capping of cell membrane components. Cell. 1979 Jan;16(1):213–222. doi: 10.1016/0092-8674(79)90202-2. [DOI] [PubMed] [Google Scholar]
- Heath J. P., Dunn G. A. Cell to substratum contacts of chick fibroblasts and their relation to the microfilament system. A correlated interference-reflexion and high-voltage electron-microscope study. J Cell Sci. 1978 Feb;29:197–212. doi: 10.1242/jcs.29.1.197. [DOI] [PubMed] [Google Scholar]
- Hynes R. O., Destree A. T. 10 nm filaments in normal and transformed cells. Cell. 1978 Jan;13(1):151–163. doi: 10.1016/0092-8674(78)90146-0. [DOI] [PubMed] [Google Scholar]
- Lazarides E., Burridge K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell. 1975 Nov;6(3):289–298. doi: 10.1016/0092-8674(75)90180-4. [DOI] [PubMed] [Google Scholar]
- Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature. 1980 Jan 17;283(5744):249–256. doi: 10.1038/283249a0. [DOI] [PubMed] [Google Scholar]
- Lloyd C. W., Smith C. G., Woods A., Rees D. A. Mechanisms of cellular adhesion. II. The interplay between adhesion, the cytoskeleton and morphology in substrate-attached cells. Exp Cell Res. 1977 Dec;110(2):427–437. doi: 10.1016/0014-4827(77)90309-3. [DOI] [PubMed] [Google Scholar]
- Pearlstein E., Dienstman S. R., Defendi V. Identification of macrophage external membrane proteins and their possible role in cell adhesion. J Cell Biol. 1978 Oct;79(1):263–267. doi: 10.1083/jcb.79.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Singer I. I. The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell. 1979 Mar;16(3):675–685. doi: 10.1016/0092-8674(79)90040-0. [DOI] [PubMed] [Google Scholar]
- Starger J. M., Brown W. E., Goldman A. E., Goldman R. D. Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells. J Cell Biol. 1978 Jul;78(1):93–109. doi: 10.1083/jcb.78.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wehland J., Osborn M., Weber K. Cell-to-substratum contacts in living cells: a direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and alpha-actinin. J Cell Sci. 1979 Jun;37:257–273. doi: 10.1242/jcs.37.1.257. [DOI] [PubMed] [Google Scholar]
- Zackroff R. V., Goldman R. D. In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6226–6230. doi: 10.1073/pnas.76.12.6226. [DOI] [PMC free article] [PubMed] [Google Scholar]