Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues

Abstract

Desmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS‐PAGE. These preparations were extracted in 9 M urea, 10 mM Tris‐HCl (pH 9), and 25 mM B‐mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea‐soluble and insoluble fractions were analyzed by SDS‐PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin‐like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris‐HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane‐associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoproteins or keratin.