Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation.
Human epidermal cells grown in culture synthesize abundant keratins. These keratins are similar to those of stratum corneum of human epidermal callus in their insolubility in dilute aqueous buffers, their molecular weight range of 40,000 to 60,000, their immunolgical reactivity, and their ability to assemble into 80 A tonofilaments in vitro; but there are differences in the molecular weights of some of the proteins, the number of components, and their charge heterogeneity, related at least in part to phosphorylation. About 30% of all the proteins of living cultured keratinocytes consists of keratins, compared with over 85% of stratum corneum. All the keratins of human stratum corneum were found to be cross-linked by intermolecular disulfide bonds while most keratins of the living cells were not. As the cells mature in Methocel-stabilized suspension culture, their keratins become increasingly disulfide cross-linked. When uncross-linked tonofilaments of living keratinocytes are dissolved in 8 M urea and the filaments reconstituted in vitro their keratins become disulfide cross-linked under aerobic conditions and consequently insoluble in solutions of 8 M urea or sodium dodecyl sulfate. The results indicate that the uncross-linked state of the keratins in living cells is due to the reducing intracellular environment and not to a precursor state related to the primary structure of the proteins. The disulfide cross-links stabilizing the keratin filaments must be distinguished from the epsilon-(gamma-glutamyl)lysine cross-links stabilizing the cornified cell envelope.
D002454:Cell Differentiation; D002478:Cells, Cultured; D003429:Cross Reactions; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D005779:Immunodiffusion; D007633:Keratins / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D012867:Skin / Q000378:metabolism*
D004220:Disulfides; D046911:Macromolecular Substances; D007633:Keratins