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.