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.
Eichner
R
R
Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Kahn
M
M
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