Harvima I T, Naukkarinen A, Harvima R J, Fräki J E
Department of Dermatology, University of Kuopio, Finland.
Arch Dermatol Res. 1988;280(6):363-70. doi: 10.1007/BF00426615.
Trypsin-like proteinase isolated from human skin was localized in cutaneous mast cells using immunoperoxidase and enzyme-histochemical techniques. Skin biopsy specimens were taken from four mastocytoma and four healthy patients. Immunoperoxidase staining was performed with protein A-sepharose purified rabbit polyclonal antibody raised against human skin tryptase and using aminoethylcarbazole as chromogen. The positively stained cells in the dermis were granular in character. Using peptide 4-methoxy-2-naphthylamide substrates (Bz-Arg-MNA, Z-Lys-Arg-MNA, Z-Gly-Arg-MNA, Z-Pro-Arg-MNA and Z-Gly-Pro-Arg-MNA) and Fast Garnet GBC as chromogen the red azo dye was found to precipitate in the cytoplasmic granules of the cutaneous mast cells. The enzymatic reaction was totally inhibited by diisopropyl fluorophosphate, leupeptin, and benzamidine. No marked inhibition was seen with soybean trypsin inhibitor and alpha-1-anti-trypsin. The best substrate was Z-Gly-Pro-Arg-MNA giving the strongest red azo dye when incubation time was 15, 30 or 60 min. These results show the localization of human skin tryptase in dermal mast cells and the usefulness of Z-Gly-Pro-Arg-MNA as a suitable substrate tested for enzyme-histochemical localization of mast cells in healthy or mastocytoma skin.
采用免疫过氧化物酶法和酶组织化学技术，对从人皮肤中分离出的类胰蛋白酶进行定位，发现其存在于皮肤肥大细胞中。取自4例肥大细胞瘤患者和4例健康人的皮肤活检标本。用蛋白A-琼脂糖纯化的兔抗人皮肤类胰蛋白酶多克隆抗体进行免疫过氧化物酶染色，以氨基乙基咔唑作为显色剂。真皮中阳性染色的细胞呈颗粒状。使用肽4-甲氧基-2-萘酰胺底物（Bz-Arg-MNA、Z-Lys-Arg-MNA、Z-Gly-Arg-MNA、Z-Pro-Arg-MNA和Z-Gly-Pro-Arg-MNA），并以固红GBC作为显色剂，发现红色偶氮染料沉淀在皮肤肥大细胞的细胞质颗粒中。酶促反应完全被二异丙基氟磷酸、亮抑酶肽和苯甲脒抑制。大豆胰蛋白酶抑制剂和α-1-抗胰蛋白酶未见明显抑制作用。最佳底物是Z-Gly-Pro-Arg-MNA，在孵育15、30或60分钟时产生最强的红色偶氮染料。这些结果表明人皮肤类胰蛋白酶在真皮肥大细胞中的定位，以及Z-Gly-Pro-Arg-MNA作为一种合适底物在健康或肥大细胞瘤皮肤中用于肥大细胞酶组织化学定位检测的实用性。
