Intracellular Activity of Lysosomal Glucosylceramidase Measured with 4-Nonylumbelliferyl β-glucoside

Abstract

Enzymatic activity of lysosomal glucosylceramidase was determined in intact murine hybridoma and macrophage cells with the synthetic substrate nonylumbelliferyl-ß-glucoside (NUG). The substrate was applied as complex with bovine serum albumin (two binding sites, K d 2.2 ± 0.3 ). The transport of the artificial substrate from medium to the enzyme was explored by measurements of substrate concentrations in cellular membranes and of endocytosis rate relative to substrate hydrolysis. The results indicated that, after enrichment in the plasma membrane, the substrate is mainly transported by simple diffusion. Release of nonylumbelliferone monitored fluorimetrically after disintegration of the cells in borate buffer containing Triton X-100 at pH 9.5 showed that 10 <s cells of both cell lines hydrolysed 1 -1.5 nmol substrate/min at a total concentration of 0.1 mM NUG in the medium. Substrate hydrolysis was prevented by preincubating the cells with conduritol B epoxide (CBE), a specific active site-directed inhibitor of lysosomal glucosylceramidase. The substrate concentration at the site of the enzyme and maximal activity were evaluated by the inhibiting effect of the substrate on the inactivation rate by conduritol epoxide. The rate of inhibitor uptake measured with bromo-[ 3 H]conduritol epoxide was shown to be not rate-limiting for the inactivation reaction. The molar concentration of the enzyme was determined by labeling with bromo-[ 3 H]conduritol epoxide. Comparison of the maximal intracellular activity with that of the enzyme after disintegration and activation by taurocholate showed a 20-fold lower activity in the native environment. The intracellular inactivation rate compared to the solubilized and activated state with CBE was 6-fold lower with the hybridoma enzyme and 22-fold lower with the macrophage enzyme. Intrazelluläre Aktivität lysosomaler Glucosylceramidase,Zusammenfassung: Die enzymatische Aktivität der lysosomalen Glucosylceramidase wurde in intakten Hybridomzellen und Makrophagen aus der Maus mit dem synthetischen Substrat Nonylumbelliferyl-ß-glucosid (NUG) bestimmt. Das Substrat wurde als Komplex mit Serumalbumin in Lösung gebracht gemessen mit 4-Nonylumbelliferyl-ß-glucosid (zwei Bindungsstellen, K d = 2.2 ± 0.3 ). Der Transport des künstlichen Substrats vom Medium zum Enzym wurde durch Messung der Substratkonzentrationen in zellulären Membranen und durch Messung der Endocytoserate relativ zur Substrathydrolyse untersucht. Die Resultate zeigten, daß das Enzymes:

Functional Organization of the Golgi Apparatus in Glycosphingolipid Biosynthesis

“…In the presence of CBE hydrolysis of [ 3 H]t-GlcCer was efficiently inhibited (Fig. 1, lane 3) (58,59). These conditions allowed the determination of the ␤-glucosidase (without added CBE) or LacCer synthase (in presence of CBE).…”

Role of pH in determining the cell-type-specific residual activity of glucocerebrosidase in type 1 Gaucher disease.

“…However, in the presence of the natural activator protein saposin C and phosphatidylserine, the enzyme functions in a near normal fashion at an appropriate acidic pH. To obtain information on the properties ofthis mutated enzyme in situ, the irreversible inhibitor CBE has been used, an approach that was developed and used previously by other investigators (25,36,37). Although it is obvious that the reaction of glucocerebrosidase with CBE involves only part of the total reaction mechanism of substrate hydrolysis (discussed in more detail in [2 ]), analysis of CBEinduced inactivation of glucocerebrosidase is informative because the reactivity of (mutant) glucocerebrosidase with CBE appears to reflect to a large extent the ability of enzyme to be active towards substrate.…”

Microspectrofluorometry and fluorescence imaging in the study of human cytopathology

“…A panel of endogenous-exogenous metabolic, organelleselective, and enzyme-selective fluorescent/fluorogenic probes was used to study differentiated, differentiating, gene-defective, and transformed cells (Bereiter-Hahn, 1976;Biberich and Legler, 1989;Del Bino et al, 1991;Dellinger et al, 1998;Gatt et al, 1996;Haugland, 1995;Johnson et al, 1997;Kohen et al, 1990Kohen et al, , 1991bKohen et al, , 1992aMalik et al, 1996;Pagano, 1991). Metabolic studies require the use of the endogenous fluorochrome [NAD(P)H] excited at 365 nm (emission recorded in the 450 -480 band) for study of baseline fluorescence and transient responses induced by microinjection of metabolites.…”

“…The organelle-selective fluorescent probes were dimethylaminostyrylpyridiniummethyl iodine (DASPMI) (Bereiter-Hahn, 1976) and rhodamine 123 (Goldstein and Korzack, 1981) (excitation at 436 nm, emission in the green) for mitochondria and NBD ceramide for the Golgi apparatus (Mangelsdorff, 1994). The fluorogenic probes for the lysosomal ␤-glucosidase were 4-methylumbeliferyl-␤-glucoside (UMBG) and 4-nonylumbelliferylglucopyranoside (UG9) (excitation at 365 nm) (Biberich and Legler, 1989;Gatt et al, 1996;Kohen et al, 1992cKohen et al, , 1993Richards-Chernicki, 1990). The fluorescent probes for the same enzyme were lissaminerhodaminedodecanoyl-glucocerebroside (LR12-GC) and pyrenedodecanoyl trihexosyl ceramide (Pl2THC) 25 (Gatt et al, 1996) .…”