Abstract
When serially cultivated human epidermal keratinocytes are placed in suspension culture they stop growing and form, beneath the plasma membrane, an insoluble envelope consisting of protein cross-linked by ε- (γ-glutamyl)lysine. The formation of envelopes in suspended cells is preceded by a sharp decline in the rate of protein synthesis, and most envelopes appear only after the average rate of protein synthesis has fallen to a very low level. If protein synthesis is reduced over 98 percent with cycloheximide or emetine at the time that surface-grown cells are placed in suspension culture, cross-linked envelopes form in most of the cells. This shows that the precursor of the envelope and the cross-linking enzyme are already in the cytoplasm in most cells of growing surface cultures. The process of envelope formation by suspension cultures is actually accelerated by the inhibitors of protein synthesis; an increased number of cells with cross-linked envelopes is observable within 4-6 h after the addition of cycloheximide. The inhibitor also induces a large fraction of the cells of surface cultures to form enveloped within a few days. These findings suggest that arrest of protein synthesis leads to activation of the cross-linking process. Agents known to inhibit transglutaminase-mediated protein cross-linking-putrescine, iodoacetamide, and ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA)- also prevent envelope formation. Though the activity of the cross-linking transglutaminase depends on the presence of cellular Ca++, we have not been able to activate the cross-linking process by high external Ca++ concentration or ionophores.
Full Text
The Full Text of this article is available as a PDF (488.7 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Buxman M. M., Wuepper K. D. Isolation, purification and characterization of bovine epidermal transglutaminase. Biochim Biophys Acta. 1976 Dec 8;452(2):356–369. doi: 10.1016/0005-2744(76)90185-6. [DOI] [PubMed] [Google Scholar]
- Folk J. E., Chung S. I. Molecular and catalytic properties of transglutaminases. Adv Enzymol Relat Areas Mol Biol. 1973;38:109–191. doi: 10.1002/9780470122839.ch3. [DOI] [PubMed] [Google Scholar]
- Goldsmith L. A., Baden H. P., Roth S. I., Colman R., Lee L., Fleming B. Vertebral epidermal transamidases. Biochim Biophys Acta. 1974 May 10;351(1):113–125. doi: 10.1016/0005-2795(74)90070-1. [DOI] [PubMed] [Google Scholar]
- Green H. Terminal differentiation of cultured human epidermal cells. Cell. 1977 Jun;11(2):405–416. doi: 10.1016/0092-8674(77)90058-7. [DOI] [PubMed] [Google Scholar]
- Lorand L., Rule N. G., Ong H. H., Furlanetto R., Jacobsen A., Downey J., Oner N., Bruner-Lorand J. Amine specificity in transpeptidation. Inhibition of fibrin cross-linking. Biochemistry. 1968 Mar;7(3):1214–1223. doi: 10.1021/bi00843a043. [DOI] [PubMed] [Google Scholar]
- Lorand L., Weissmann L. B., Epel D. L., Bruner-Lorand J. Role of the intrinsic transglutaminase in the Ca2+-mediated crosslinking of erythrocyte proteins. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4479–4481. doi: 10.1073/pnas.73.12.4479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pisano J. J., Finlayson J. S., Peyton M. P. Chemical and enzymic detection of protein cross-links. Measurement of epsilon-(gamma-glutamyl)lysine in fibrin polymerized by factor XIII. Biochemistry. 1969 Mar;8(3):871–876. doi: 10.1021/bi00831a016. [DOI] [PubMed] [Google Scholar]
- Reed P. W., Lardy H. A. A23187: a divalent cation ionophore. J Biol Chem. 1972 Nov 10;247(21):6970–6977. [PubMed] [Google Scholar]
- Rice R. H., Green H. The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein. Cell. 1977 Jun;11(2):417–422. doi: 10.1016/0092-8674(77)90059-9. [DOI] [PubMed] [Google Scholar]
- Stanners C. P., Becker H. Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. I. Ribosome function. J Cell Physiol. 1971 Feb;77(1):31–42. doi: 10.1002/jcp.1040770105. [DOI] [PubMed] [Google Scholar]
- Sun T. T., Green H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope. Cell. 1976 Dec;9(4 Pt 1):511–521. doi: 10.1016/0092-8674(76)90033-7. [DOI] [PubMed] [Google Scholar]
- Warner J. R., Girard M., Latham H., Darnell J. E. Ribosome formation in HeLa cells in the absence of protein synthesis. J Mol Biol. 1966 Aug;19(2):373–382. doi: 10.1016/s0022-2836(66)80011-6. [DOI] [PubMed] [Google Scholar]