Intralysosomal hydrolysis of glycyl-l-phenylalanine 2-naphthylamide

Abstract

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.

NAADP Mobilizes Calcium from the Endoplasmic Reticular Ca2+ Store in T-lymphocytes

“…To analyze whether in Jurkat T-lymphocytes lysosomes represent a Ca 2ϩ store sensitive to NAADP, we treated intact cells with GPN, a substrate of cathepsin C. Via osmotic lysis, the metabolic products of GPN selectively disrupt lysosomal membranes (11,27). The resulting significant loss of lysosome staining by LysoTracker Red was confirmed in Jurkat T cells (Fig.…”

Microglial migration mediated by ATP-induced ATP release from lysosomes

“…To directly determine whether lysosomes are involved in microglial chemotaxis, we incubated cultures with glycylphenylalanine-2-naphthylamide (GPN), a substrate of the lysosomal exopeptidase cathepsin C that selectively induces lysosome osmodialysis [16,27]. We found that the cell mobility (data not shown) and ATPγS-induced migration ( Figure 7A) were reversibly blocked in microglia treated with 100 µM GPN, suggesting a crucial role of lysosomes in microglial migration.…”

Effect on lysosomes of invertase endocytosed by rat-liver

“…Enzymes were assayed according to the following references: invertase [6], cathepsin C [7], /?-galactosidase and N-acetylglucosaminidase [8], acid phosphatase [9], acid deoxyribonuclease [lo], arylsulfatase [ll].…”