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The extraction and characterization of bovine epidermal alpha-keratin.

ABSTRACT: 1. The alpha-fibrous protein (alpha-keratin) component of bovine epidermis has been extracted and characterized. 2. Prekeratin, a multichain unit of the epidermal tonofilaments, was shown to consist of six different polypeptide chains on polyacrylamide-gel systems containing sodium dodecyl sulphate or sodium decyl sulphate with discontinuous gel buffers, but only three chains were seen when a gel system containing sodium dodecyl sulphate with a continuous gel buffer was used. 3. Extraction of the 'keratinized' stratum corneum and the living part of the epidermis with urea buffers at pH 7.6 or 9.0 released 60% of the total dry weight of the tissues in the form of alpha-helical polypeptides. 4. The numbers, relative amounts and properties of the extracted polypeptides were the same as the subunits of prekeratin and thus are derived from the tonofilaments in situ. 5. The subunits of prekeratin and the polypeptides extracted from the living cell layers contained an average of six cysteine residues, but those from the stratum corneum contained an average of three intrachain disulphide bonds. 6. The polypeptide chains aggregated through non-covalent interactions in vitro into filaments that were similar to the tonofilaments. 7. Since the polypeptides could be released from the stratum corneum without breaking covalent bonds, it is concluded that such bonds do not cross-link the tonofilaments and non-fibrous keratohyalin. It is suggested that the tonofilaments and keratohyalin of bovine epidermis are associated by secondary bonding forces.

SUBMITTER: Steinert PM

PROVIDER: S-EPMC1165590 | biostudies-other | 1975 Jul

REPOSITORIES: biostudies-other

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Project description:To obtain a separation of the epidermal and dermal compartments in order to examine compartment specific biological mechanisms in the skin we incubated 4 mm human skin punch biopsies in ammonium thiocyanate (NH4SCN). We wanted to test 1) the histological quality of the dermo-epidermal separation obtained by different incubation times 2) the amount and quality of extractable epidermal RNA, and 3) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30 minutes incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally) but varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately. Upper buttock skin in 4 healthy subjects was exposed to sodium lauryl sulphate, or sampled directly. For each subject, 4 biopsies were obtained: Two from inflamed skin, and two from adjacent normal skin. One irritated and one normal skin sample was placed directly in RNAlater. The remaining two samples were incubated in ammonium thiocyanate for 30 minutes at RT and then placed in RNAlater without performing any separation of the dermal and epidermal layers. This was done to investigate the effect of 30 minutes treatment with ammonium thiocyanate on both inflamed and non-inflamed skin. Data was normalized with quantile method (matrix 1) Forearm biopsies from 13 volunteers were separated to epidermis and dermis by use of ammonium thiocyanate. For comparison of full skin and epidermis without irritation, data from identical probe sets from HG_U133A 2.0 and HG_U133 plus 2.0 was extracted and normalised as one data set using quantile method (matrix 2).

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The extraction and characterization of bovine epidermal alpha-keratin.

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