Differential extraction of keratin subunits and filaments from normal human epidermis.
We have investigated keratin interactions in vivo by sequentially extracting water-insoluble proteins from normal human epidermis with increasing concentrations of urea (2, 4, 6, and 9.5 M) and examining each extract by one- and two-dimensional gel electrophoresis, immunoblot analysis using monoclonal anti-keratin antibodies, and EM. The viable layers of normal human epidermis contain keratins K1, K2, K5, K10/11, K14, and K15, which are sequentially expressed during the course of epidermal differentiation. Only keratins K5, K14, and K15, which are synthesized by epidermal basal cells, were solubilized in 2 M urea. Extraction of keratins K1, K2, and K10/11, which are expressed only in differentiating suprabasal cells, required 4-6 M urea. Negative staining of the 2-M urea extract revealed predominantly keratin filament subunits, whereas abundant intermediate-sized filaments were observed in the 4-urea and 6-M urea extracts. These results indicate that in normal human epidermis, keratins K5, K14, and K15 are more soluble than the differentiation-specific keratins K1, K2, and K10/11. This finding suggests that native keratin filaments of different polypeptide composition have differing properties, despite their similar morphology. Furthermore, the observation of stable filaments in 4 and 6 M urea suggests that epidermal keratins K1, K2, and K10/11, which ultimately form the bulk of the protective, nonviable stratum corneum, may comprise filaments that are unusually resistant to denaturation.
Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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Eichner
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Kahn
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000911:Antibodies, Monoclonal; D003599:Cytoskeleton / Q000648:ultrastructure*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007633:Keratins / Q000302:isolation & purification* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D012016:Reference Values; D012867:Skin / Q000032:analysis* / Q000648:ultrastructure; D012995:Solubility; D014508:Urea
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D007633:Keratins; D014508:Urea