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 noncornified 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.
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, t7% carbohydrate, and 10% lipid. Nonpolar amino acids predominate in desmosomal protein, representing 456 residues per 1,000. The sialic acid content is 5 n M / m g 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% /5-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.
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.
The purpose of this study has been to obtain information on the dcvclopment of the envclop of horny cells that resists the action of keratinolytic agents. Toward this end the epidcrmis, oral mucosa, and tongue epithclium of various vcrtcbratcs, as well as the isolated envelopes of horny cclls, were examined by electron microscopy. It was found that small cytoplasmic granulcs (l,000 to 5,000 A) that devclop within differentiating epithelial cclls 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.