Reiko Hirai

The Synthesis of the Cystine-Rich Proteins in Rat Epidermis: I. Analysis by [35S]Cystine Incorporation

Three-day-old Sprague-Dawley rats were injected intraperitoneally with 100 muCi of [35S]cystine. They were then sacrificed at 30 minutes, 2, 4, and 10 hours. The skins were epilated by the beeswax-resin procedure and the epidermis was separated by soaking in NH4Cl solution. Neither hair with hair follicles nor hair sheath was attached to the epidermal sheets after the epilation. Keratohyalin granules were solubilized after the freezing and thawing procedure. Living cell layers were separated from stratum corneum following further incubation in NH4Cl solution. Both the living cell layers and the stratum corneum were extracted with 8 M alkaline urea. Seventy-two to 92% of the total radioactivity incorporated into epidermal proteins was found in the urea-insoluble fraction of the stratum corneum. Only 2.4 to 6.4% of the total radioactivity was incorporated into the keratohyalin granular fraction. The specific radioactivity which was incorporated into the urea-insoluble fraction of the stratum corneum was still increasing 10 hours after injection, though the radioactivity in other fractions was decreasing at 10 hours following injection. The highest specific radioactivity of [35S]cystine was found in the urea-soluble fraction of the living cell layers, and was being rapidly turned over.