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Keratin was extracted from normal human horny cells of the leg, calluses of the sole, and psoriatic scales. After dissociation in sodium dodecyl sulfate the polypeptides were separated by Laemmli's gel electrophoresis method and their molecular weights and relative amounts determined. Normal horny cells contained 3 polypeptide chains of Mr 67K, 59K, and 57K, while those of callus contained 9 polypeptides of Mr 67K, 66K, 63K, 62K, 58K, 54K, 52K, 48K, and 45K. In both cases all keratin polypeptides participated in filament reassembly in vitro and were recovered from the filaments. In psoriatic scale keratin, 7 prominent polypeptides were detected having Mr 67K, 59K, 57K, 50K, 48K, 42K, and 40K. The 67K polypeptide could not be recovered from reassembled filaments. Ultrastructural studies revealed that these filaments are imperfect and readily aggregate into thick fibrils. These observations indicate that there are significant differences in composition of keratin of normal horny cells, calluses, and psoriatic scales.

Prekeratin was isolated from bovine snout epidermis with 0.1 M citric acid/sodium citrate buffer, pH 2.6 (buffer A). Filaments, 6.0-9.0 nm wide, were produced by dialysis against low ionic strength buffer A or by dissociating prekeratin in 8 M urea solution followed by dialysis against 0.005 M Tris-HCl buffer, pH 8.0. The polypeptide composition of both prekeratin and filaments was studied by four different SDS-polyacrylamide gel electrophoresis methods. The best resolution was obtained by Laemmli's technique in which both prekeratin and filaments were separated into three major and seven distinct minor bands of polypeptides. The major ones comprise approx. 70% of total polypeptides and their estimated molecular weights are 68 000, 54 000, and 50 000. The molecular weight of minor ones is in decreasing order 65 000, 63 000, 61 000, 58 000, 47 000, 44 000 and 42 000. It is proposed that the major polypeptides form the backbone structure of epidermal filaments and the minor polypeptides play a role in its stabilization.

Components of desmosomes, filaments, and keratohyaline granules were studied by electron microscope and biochemical methods to clarify their role in the stabilization and keratinization of the epidermis. Isolated desmosomes are composed of 76% protein, 17% carbohydrate, and 10% lipid. The bulk of protein consists of a "spectrin"-like fibrous protein, presumably present in the plaque, and of glycoproteins in the desmosomal interspace. The main component of filaments, prekeratin, is a low-sulfur alpha-protein composed of a pair of three-chain subunits with non-alpha-helical segments separated by 200 A-long alpha-helical regions. The major component of isolated keratohyaline granules, the amorphous particulate material, is formed by a high-sulfur protein with a single-type of polypeptide chain. Polypeptide chains comparable to those found in prekeratin and keratohyaline granules were recovered from extracts of horny cells. Within the living part of the epidermis, filaments hypothetically form a cytoskeletal system which is anchored to desmosomes by a filamentous plaque protein. Glycoproteins are involved in the formation of strong junctions between the cells which enable the living part of the epidermis to respond as a whole to mechanical stress. The stratum corneum is stabilized by a similar system in a consolidated state which is less extensible. Horny cells are enveloped by a thickened membrane and the interfilamentous spaces are filled with various proteins including the sulfur-rich amorphous protein found in keratohyaline granules.

Early studies have already shown that the tonofibrils of malpighian cells consist of a --SH containing fibrous alpha-protein. It was assumed that the highly resistant protective substance, keratin, was formed by the conversion of --SH groups into --S--S--bonds in this protein. This chemical reaction was regarded as the most significant event of the keratinization process. Recent studies show that keratinization proceeds by a synthetic and a degradative stage and that ultimately a complex protective substance is formed. Horny cells become filled with --SH-containing filaments embedded in a --S--S---rich amorphous matrix. This complex is encased by a thickened membrane rendered insoluble by --S--S bonds and an unknown, highly resistant bond. In the stratum corneum, the intercellular space is occupied by bipolar lipids originating from the discharged lamellae of membrane-coating granules.

A method is reported for the isolation of desmosomes in a high yield and of a purity suitable for biochemical analysis. The procedure utilizes the selective solubilizing action of citric acid-sodium citrate (CASC) buffer, pH 2.6, on the non-cornified layers of cow nose epidermis, followed by discontinuous sucrose density gradient centrifugation. Electron microscopy with both thin sections of pellets and unfixed spread preparations reveals that after centrifugation, desmosomes are located mainly at the 55-60% sucrose interface. In the desmosome preparation thus obtained, the characteristic desmosome structure is well preserved, showing the midline, unit membranes, and dense plaques. Furthermore, removal of the epidermal filament bundles by the solubilizing action of CASC buffer has revealed a finely filamentous layer on the cytoplasmic surface of the plaques. The dimensions, location, and appearance of this layer correspond with those of the "connecting component" which has been previously suggested as being responsible for the attachment of epidermal filament bundles to the desmosome.

The structure of single filaments of horny cells of normal and psoriatic epidermis was studied in the electron microscopy in situ and in filaments reconstituted from urea extracts by dialysis, against low ionic strength buffer. It was found that both in situ and reconstituted filaments consist of 20 A protofibrils. In filaments of normal horny cells the protofibrils seem to form a rope-like structure while in those of psoriatic horny cells protofibrils appear randomly arranged. The filaments reconstituted from extracts of psoriatic scales range in width from 90 to 500 A, indicating a defect in lateral assembly of protofibrils. Numerous small particles are detectable at high magnification in extracts of both normal and psoriatic horny cells, which are about 20 A wide and 200 A long. These particles are consisted to be the basic structural units from which the protofibrils are formed, by end-to-end junctions.

Desmosomes, isolated from cow nose epidermis by a method utilizing citrate buffer pH 2.6 and density gradient centrifugation, have been analyzed and found to contain approximately 76% protein, 17% carbohydrate, and 10% lipid. Nonpolar amino acids predominate in desmosomal protein, representing 456 residues per 1,000. The sialic acid content is 5 nM/mg of protein. The lipid fraction is composed of approximately 40% cholesterol and 60% phospholipids. Desmosomes are completely solubilized by incubation with 2% sodium dodecyl sulphate and 1% beta-mercaptoethanol. Gel electrophoresis of the denatured desmosomal proteins reveals 24 bands, with mobilities corresponding to a molecular weight range of 15,000-230,000 daltons. Seven of these are considered to be major bands, together constituting 81% of the desmosomal protein. Bands 1 and 2, of molecular weights 230,000 and 210,000 daltons, together comprise 28% by weight of the desmosome. It is suggested that these protein chains are located in the desmosomal plaque. Bands 3 and 4 are PAS-positive, constitute 23% of the desmosomal protein, and have apparent molecular weights of 140,000 and 120,000 daltons, respectively. At least part of this material must originate from the carbohydrate-containing layer which is demonstrated, by histochemistry, to be present in the desmosomal interspace. The possible nature and origin of the remaining major bands, of molecular weights 90,000, 75,000, and 60,000 daltons, are discussed.

Pulverized cornified epithelium of human skin was divided into a "soluble fraction" and a "residue." About half of the "soluble fraction" proved to be soluble epidermal keratin (keratin A); the remainder, dialyzable substances of low molecular weight. The "residue" contained epidermal keratin and resistant cell membranes of cornified cells. Epidermal keratin was found to form an oriented and dense submicroscopic structure in the cornified cells. It showed high resistance toward strong acid and moderately strong alkali solutions as well as concentrated urea. In strong alkali, reducing substances, alkaline urea, and mixtures of reducing substance with alkali, epidermal keratin dissociated and yielded a non-dialyzable derivative of high molecular weight (keratin B) which resembled true proteins. The cell membranes of cornified cells showed higher resistance toward strong alkali and reducing substance than did epidermal keratin.

The purpose of this study has been to obtain information on the development of the envelop of horny cells that resists the action of keratinolytic agents. Toward this end the epidermis, oral mucosa, and tongue epithelium of various vertebrates, as well as the isolated envelopes of horny cells, were examined by electron microscopy. It was found that small cytoplasmic granules (1,000 to 5,000 A) that develop within differentiating epithelial cells move toward the cell periphery, and after fusion with the plasma membrane, empty their contents into the intercellular spaces. The content of the granules spreads over the cell surfaces, and subsequently a thickened and coated cell envelope is formed that resists the action of keratinolytic agent. The membrane-coating granule is regarded as a specific differentiation product of the keratinizing epithelium. It contains numerous inner membranes and is assumed to engage in synthetic activities such as, perhaps, the formation of polysaccharides.

To explore the submicroscopic structure of the human callus by the polarization optical method, serial sections were prepared in three principal planes and the sign of double refraction as well as optic axes of various regions of the sections was determined. It was concluded that in areas under the grooves keratin is oriented parallel to the surface plane of the callus and to the direction of the grooves. In component structures of ridge areas such as in sweat duct areas a circumferential, in cross-band areas a linear, and in other parts a random structure was seen. The cross-bands of ridge areas in association with groove areas revealed a regular lattice-like submicroscopic pattern which was considered as an important mechanical system of the cornified epithelium.

Keratohyalin granules were isolated in the native form from the epidermis of newborn rats by the use of citric acid and a detergent. The isolated granules revealed a fine granular substructure in the electron microscope similar to that seen in situ. Analyses of amino acids by automated column-chromatography showed that proline and cystine are present in large proportions whereas histidine is present in a small amount. Accordingly, it was concluded that keratohyalin represents a sulfur-rich amorphous precursor of the horny cell content, rather than a sulfur-poor side product of the keratinization process, or a unique histidine-rich protein as proposed by in situ histochemical and radioautographic studies.

The epidermis of adult Rana pipiens has been studied by electron microscopy and histological and histochemical methods. It was found that the epidermis is engaged in the production of both keratin and mucus. The basal cells are mainly filled with tonofilaments, whereas the cells located in the mid-portion of the epidermis contain both tonofilaments and mucous granules. Golgi vesicles and endoplasmic reticulum are found in relative abundance in the mucus-producing cells and seem to be involved in the production of mucous granules. The mucus seen was partly retained within the cells and partly secreted into the intercellular spaces. The outermost keratinized cells contain mainly filaments and a few remnants of cell constituents.

Epithelial cells changing from the granular stage of differentiation to the horny stage are more numerous, and reveal sequential events of transformation in finer detail in the rumen epithelium than in other keratinizing epithelia thus far studied in the electron microscope. Studies of such cells indicate that transformation is initiated by the release of hydrolytic enzymes, as evidenced by the appearance of lysosomes. As lysosomes increase in number, the nucleus, ribosomes, mitochondria, Golgi apparatus, and mucous granules are gradually degraded. Furthermore, marked changes occur in permeability of the plasma membrane as voluminous amounts of the lysed cell components pass through and accumulate in the intercellular space in the form of an amorphous mass. Filaments, keratohyalin granules, and the content of the ER (ER-protein) are not lysed, revealing the action of released enzymes to be specific. During transformation, filaments become displaced toward the cell periphery and keratohyalin granules disperse and mix with the ER-protein in the cell center. Subsequently, the keratohyalin-ER-protein complex infiltrates the filament network whereby a fibrous-amorphous cell content is formed. Loss of fluids through the plasma membrane leads to reduction of cell volume and consolidation of the remaining cell content. The deep interdigitations formed between the cells ultimately interlock the outer part of the epithelium into a cohesive and protective stratum corneum.