The 55-kd keratohyalin granule protein has the same epitope as the 43-kd stratum corneum membrane protein: immunofluorescence and immunoblotting studies using a monoclonal antibody to the 55-kd keratohyalin granule protein.
A monoclonal antibody (Ted-R-1) to the keratohyalin granules (KHGs) of the newborn rat epidermis was developed by immunizing mice with a pH 4.7 hematoxylin-stainable fraction of the extract in a 50 mM Tris-HCl buffer, pH 7.3, containing 10 micrograms/ml phenylmethylsulfonyl fluoride (PMSF) from the newborn rat whole epidermis. Using an indirect immunofluorescence technique, Ted-R-1 was located in two places: (a) the KHGs and (b) the cell membrane region of the stratum corneum. Under immunoelectron microscopy, the antigenic materials were located at the periphery of the KHGs. Immunoblotting analyses of the antigenic pH 4.7 fraction, the Tris-HCl extract from the stratum corneum, and the Tris-8M urea extract from freeze-dried whole skin demonstrated that there were 55 kd, 43 kd, and 78 kd polypeptides, respectively, with which Ted-R-1 reacts. No positive spot was seen in the dermal extract. The major amino acids of the 55 kd protein were glycine (24.4%), serine (17.3%), glutamic acid (14.1%), and alanine (7.7%), while the histidine residue was only 3.1%. On the basis of its amino acid composition, the 55 kd proteins is the third component of KHGs - in addition to histidine-rich and cystine-rich proteins.
Tezuka
T
T
Department of Dermatology, Kinki University School of Medicine, Osaka, Japan.
Takahashi
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M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002846:Chromatography, Affinity; D004817:Epidermis / Q000032:analysis* / Q000648:ultrastructure; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006416:Hematoxylin; D015151:Immunoblotting / Q000379:methods; D007525:Isoelectric Focusing; D007633:Keratins / Q000032:analysis* / Q000276:immunology; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008565:Membrane Proteins; C001500:keratohyalin; D007633:Keratins; D006416:Hematoxylin