The Substrate of Epidermal Proteases

In newborn rat skin, disulfide bonds exist in the cell membrane of the stratum corneum and in the cytoplasm of the stratum granulosum. However, in the latter region, they can be detected after ethanol fixed epidermis remains for 2 weeks at 4°C. Thus, the idea arose that the visualization of the disulfide bonds in the stratum granulosum may be caused by proteases. Therefore, the in situ effects by both activation and inhibition of epidermal proteases were examined in tissue sections. Cryostat sections which were fixed in cold ethanol (—20°C, 98%) were incubated either in PBS, pH 7.4, containing 10mM 2‐ME as a control or in an epidermal homogenate (15,000×g supernatant fraction containing 2‐ME) at 37°C for 30 minutes and 4 hours, respectively, in order to activate the epidermal proteases and then reacted with NEM, 2‐ME, and DACM in steps. Many minute fluorescent particles were seen in the stratum granulosum under both of the above conditions. However, with incubation in PBS without 2‐ME, no fluorescent particles were seen. In addition, when the tissue was treated with NEM and zinc chloride before incubation, no fluorescent particles were seen. O‐phenanthrolin remarkably inhibited the appearance of these fluorescent particles. In contrast, leupeptin and antipain only slightly inhibited the appearance of these fluorescent particles, and pepstatin and phenylmethylsulfonyl fluoride didn't inhibit at all. In general, as NEM and zinc chloride strongly inhibit thiol proteases, these findings suggest that presence of the minute fluorescent particles in the stratum granulosum could be due to the effects of proteases, especially thiol proteases.