Document Detail


Use of glycyl-L-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles.
MedLine Citation:
PMID:  8198538     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
T O Berg; E Strømhaug; T Løvdal; O Seglen; T Berg
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  300 ( Pt 1)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1994 May 
Date Detail:
Created Date:  1994-06-28     Completed Date:  1994-06-28     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  229-36     Citation Subset:  IM    
Affiliation:
Department of Tissue Culture, Norwegian Radium Hospital, Montebello, Oslo.
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MeSH Terms
Descriptor/Qualifier:
Animals
Asialoglycoproteins / metabolism
Cathepsin C
Cellobiose / metabolism
Dipeptides / metabolism,  pharmacology*
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / metabolism*
Endocytosis*
Lysosomes / drug effects*,  metabolism
Male
Metrizamide / chemistry
Orosomucoid / analogs & derivatives,  metabolism
Rats
Rats, Wistar
Subcellular Fractions
Substrate Specificity
Vacuoles / drug effects*,  metabolism
Vinblastine / pharmacology
Chemical
Reg. No./Substance:
0/Asialoglycoproteins; 0/Dipeptides; 0/Orosomucoid; 0/asialoorosomucoid; 16462-44-5/Cellobiose; 21438-66-4/glycylphenylalanine 2-naphthylamide; 31112-62-6/Metrizamide; 865-21-4/Vinblastine; EC 3.4.14.-/Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; EC 3.4.14.1/Cathepsin C
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