Skin proteasomes (high-molecular-weight protease): purification, enzymologic properties, gross structure, and tissue...

Proteasomes (high-molecular-weight protease) were purified from rat skin, and their enzymologic properties, gross structure, and tissue distribution were investigated. Skin proteasomes were purified by successive (NH4)2SO4 fractionation and by phenyl Sepharose CL-4B and HPLC gel filtration chromatography. The molecular weights of the proteasomes were estimated from gel filtration to be 750 kD. On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the purified enzymes dissociated into several bands, the majority falling into the range of 36-20 kD. Two-dimensional electrophoretic analysis demonstrated approximately 10-15 separate protein spots with pl values varying between 3 and 10. As analyzed by electron microscopy, the gross structure of the enzymes showed an almost symmetrical ring-shaped particle with a small hole in the center. Succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide, a fluorogenic substrate for serine proteinases, demonstrated the highest activity in terms of substrate specificity. Sodium dodecylsulfate, Ca++, and some free fatty acids activated enzyme activity. Activity was inhibited by diisopropylfluorophosphate, leupeptin, N-ethylmaleimide, iodoacetamide, and chymostatin. These results show that both serine and cysteine residues are related to the enzyme activity of proteasomes. Total and specific enzyme activities in the epidermis were, respectively, 10 and 20 times higher than in the dermis. Immunohistochemical studies utilizing the avidin-biotin complex method with monoclonal antibody revealed that the enzyme is distributed throughout the epidermis. These findings indicate the epidermal localization of proteasomes.