New findings on epidermal transglutaminase substrates

Recent studies suggest the presence of natural substrates for epidermal transglutaminase in buffer extracts of epidermis. We have investigated a soluble substrate of 36,000 molecular weight present in neutral buffered extracts of cow snout epidermis. When purified to homogeneity, this protein was cross-linked by transglutaminase with 70-80% efficiency into a series of high molecular weight soluble polymers and a highly insoluble protein aggregate. The epsilon-(gamma-glutamyl) lysine bond was present in both high molecular weight soluble polymers and insoluble aggregate, but was absent from the initial 36,000 molecular weight protein. Substrate cross-linking was completely inhibited by EDTA, iodoacetamide, and 25 mM putrescine. When subjected to electrophoresis in gels containing sodium dodecyl sulfate (SDS), 90% of the 36,000 molecular weight protein was reduced to subunits of 8,000-10,000 molecular weight. We postulate intracellular synthesis of the 36,000 molecular weight initial substrate molecule, followed by insolubilization and cross-linking at the cell membrane.