Two-dimensional analysis of proteins of rat oral epithelia and epidermis

Abstract

They were separated by sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis, then treated with cyanogen bromide to cleave proteins at methionine residues, prior to re-electrophoresis at 90 ° to the first dimension. Because the histidine-rich basic protein, filaggrin, and its precursor lack methionine, they are stable in the presence of cyanogen bromide. After electrophoresis in the second dimension, these proteins lie on a diagonal in positions corresponding to their mol. wt. Filaggrin is readily identified in extracts of epidermis and newborn palate as the cyanogen bromide-stable protein of M r 44,000. The adult tongue extract had a faint spot at this position, but adult palatal and buccal extracts did not. Filaggrin had the same mol. wt in different epithelia but the amount varied with the tissue, and with age of animal. All of the oral extracts contained cyanogen bromide-stable proteins of high mol. wt that may be the precursor form of filaggrin. The keratins, which contain methionine, are cleaved by cyanogen bromide and form distinct peptide patterns below the diagonal after electrophoresis in the second dimension. The mol. wt of the major epidermal keratins were 64, 60 and 52K, those of palate 58, 54, 49 and 44.5K and those of tongue and buccal extracts, 56, 46 and 43K. Age-related changes were evident in the proportion of individual keratins in the palatal extracts. The peptide patterns generated from tongue and buccal epithelial extracts were similar, suggesting that the distribution of methionine residues in the major keratins of these two epithelia is identical. Peptides from palatal and epidermal keratins differed from each other and from those of the tongue or buccal keratins. These patterns indicate differences in primary structure and suggest that the expression of the keratins of different mol. wt in different epithelia is controlled at the genetic level.