Intralysosomal hydrolysis of glycyl-L-phenylalanine 2-naphthylamide.
Glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap), a cathepsin C substrate, induces an increase of the free and unsedimentable activities of this enzyme when incubated with a total mitochondrial fraction of rat liver. 1 mM-ZnSO4 considerably inhibits the cathepsin C total activity, measured with Gly-L-Phe-2-NNap as the substrate, in the presence of Triton X-100. The inhibition is markedly less pronounced when the free activity is determined; a high activity remains that depends on the integrity of the lysosomes; it decreases as the free activity of N-acetylglucosaminidase increases when lysosomes are subjected to treatments able to disrupt their membrane. Cathepsin C activity is reduced when thioethylamine hydrochloride is omitted from the incubation medium. Under these conditions at 37 degrees C, the free activity equals the total activity, although the lysosomes are intact, as indicated by the low free activity of N-acetylglucosaminidase. 1 mM-ZnSO4 strikingly inhibits the total activity, whereas more than 80% of the free activity remains. These observations are presented as evidence that Gly-L-Phe-2-NNap can possibly cause a disruption of the lysosomes as a result of its hydrolysis inside these organelles. In the presence of ZnSO4, intralysosomal hydrolysis becomes apparent, owing to a preferential inhibition by Zn2+ of extralysosomal hydrolysis; in the absence of thioethylamine hydrochloride, it is measurable because the disruption of lysosomes by Gly-L-Phe-2-NNap is delayed as a result of a slow-down of the reaction. The usefulness of Gly-L-Phe-2-NNap and related dipeptidyl naphthylamides in lysosomal-membrane-permeability studies is emphasized.
D000818:Animals; D002403:Cathepsins / Q000378:metabolism; D003543:Cysteamine / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013431:Sulfates / Q000494:pharmacology; D013696:Temperature; D015032:Zinc / Q000494:pharmacology; D019287:Zinc Sulfate
D004151:Dipeptides; D013431:Sulfates; C041865:glycylphenylalanine 2-naphthylamide; D003543:Cysteamine; D019287:Zinc Sulfate; D002403:Cathepsins; D015032:Zinc