Original method for the histochemical demonstration of tripeptidyl aminopeptidase I

The original histochemical method for the visualization of tripeptidyl amin opeptidase I (TPP I, EC 3.4. 14.9) is developed. The method is based on the new synthetic substrates Gly-L-Pro-L-Met(or L-Ala)-9-chloro-1-anthraquinon yl hydrazide (Gly-Pro-Met[Ala]-CAH). The final reaction product is represen ted as tripeptidyl-5-cloro-1 anthraquinonyl hydrazides (TPP-CAH). Upon the enzyme action the practically in aqeous media insoluble brown-reddish dye 5 -cloro-1-anthraquinonyl hydrazine (CAH) is released, which reacts simultane ously with aromatic aldehydes as e.g. 4-anisaldehyde (p-AA) or 4-nitrobenza ldehyde (p-NBA), resulting in microcrystalline or amorphous deeply colored hydrazones. The last compounds mark accurately the locations of enzymatic a ctivity. The biochemically suggested lysosomal localization of this collage n-degrading exopeptidase is thus confirmed on tissue sections. More informa tion about the distribution of TPP I in different rat organs is presented a s well.