The substrate was purified by DEAE-Sepharose CL-6B ion exchange chromatography, Pevikon block electrophoresis, and ACA-54 gel filtration chromatography. This modified purification procedure resulted in a 19% substrate yield. Amino acid analysis of the purified substrate was performed following treatment in SDS, alkylation in 4-vinyl pyridine, and hydrolysis with methane sulfonic acid. Results included significant levels oflysine and glutamic acid, low levels of cystine and methionine, and a

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... amount of an amino acid eluting in identical position with authentic citrulline. Studies of the sedimentation equilibrium of the substrate showed a monodispersed protein with a molecular weight of 36,780 ± 350. Ultrastructural studies of insoluble high molecular weight aggregates of the substrate revealed globular, amorphous masses lacking fibrillar structure. Using Ouchterlony double diffusion techniques, the initial substrate (36,000 m.w. substrate) and the soluble crosslinked high molecular weight substrate were shown to be immunologically identical. Indirect immunofluorescence was performed on freeze-dried sections of bovine snout epidermis using substrate specific rabbit antiserum and fluorescein-conjugated goat anti-rabbit lgG. Fluorescence was seen throughout the cytoplasm and inner cell membrane of granular cells, but was limited to the inner membrane of stratum corneum cells. Epidermal transglutaminase substrate is an amorphous, globular, citrulline containing protein s~the sized in the cytosol of keratinizing cells with ultimate localization in the cell membrane. Cells of the epidermal granular layer contain the enzyme e pidermal transglutaminase which catalyzes the formation of intermolecular e-(y-glutamyl) lysine bonds in structural proteins [1] [2] [3] [4] [5] . Protein aggregates. containing these bonds have been identified in urea-thiol extracts of human epidermis [6] and were isolated from highly insoluble prote in lining the inner membrane of keratinocytes in cell culture [7] . Recently we described the isolation and purification of a specific substrate for epidermal transglutaminase, a calcium dep endent enzyme which mediates e-(y-glutamyl) lysine crosslinking in the granular layer of epidermis [8] . This substrate was soluble in buffer extracts of bovine snout epidermis and was identified using antiserum to isolated high molecular weight substrate proteins recovered after crosslinking by the enzyme.