Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal 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 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, 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 radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.
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May 1994
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Research Article|
May 15 1994
Use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles
T O Berg;
T O Berg
*Department of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway,
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P E Strømhaug;
P E Strømhaug
*Department of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway,
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T Løvdal;
T Løvdal
†Division of Molecular Cell Biology, University of Oslo, P.O. Box 1050, Blindern, 0316 Oslo, Norway
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P O Seglen;
P O Seglen
*Department of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway,
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T Berg
T Berg
†Division of Molecular Cell Biology, University of Oslo, P.O. Box 1050, Blindern, 0316 Oslo, Norway
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Biochem J (1994) 300 (1): 229–236.
Citation
T O Berg, P E Strømhaug, T Løvdal, P O Seglen, T Berg; Use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles. Biochem J 15 May 1994; 300 (1): 229–236. doi: https://doi.org/10.1042/bj3000229
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