USE OF GLYCYL-L-PHENYLALANINE 2-NAPHTHYLAMIDE, A LYSOSOME-DISRUPTING CATHEPSIN-C SUBSTRATE, TO DISTINGUISH BETWEEN LYSOSOMES AND PRELYSOSOMAL ENDOCYTIC VACUOLES

Lysosome-disrupting enzyme substrates have been used to distinguish be tween lysosomal and prelysosomal compartments along the endocytic path way in isolated rat hepatocytes. The cells were incubated for various periods of time with I-125-labelled tyramine cellobiose (I-125-TC) cov alently coupled to asialoorosomucoid (AOM) (I-125-TC-AOM); this molecu le is internalized by receptor-mediated endocytosis and degraded in ly sosomes, where the degradation products (acid-soluble, radiolabelled s hort peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN ) and methionine O-methyl ester (MOM), which are hydrolysed by lysosom al cathepsin C and a lysosomal esterase respectively, both diffused in to hepatocytic lysosomes after electrodisruption of the cells. Intraly sosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from I- 125-TC-AOM degradation. The acid-soluble radioactivity coincided in su crose-density gradients with a major peak of the lysosomal marker enzy me acid phosphatase at 1.18 g/ml; in addition a minor, presumably endo somal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble r adioactivity, presumably in endosomes, banded (after Ih of I-125-TC-AO M uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in suc rose gradients, and was not significantly released by GPN. GPN thus ap pears to be an excellent tool by which to distinguish between endosome s and lysosomes. MOM, on the other hand, released some radioactivity a nd acid phosphatase from endosomes as well as from lysosomes.