Enzymatic activity of lysosomal glucosylceramidase was determined in intact murine hybridoma and macrophage cells with the synthetic substrate nonylumbelliferyl-β-glucoside (NUG). The substrate was applied as complex with bovine serum albumin (two binding sites, Kd 2.2 ± 0.3μM). 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 108 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 B epoxide. The rate of inhibitor uptake measured with bromo-[3H]conduritol B epoxide was shown to be not rate-limiting for the inactivation reaction. The molar concentration of the enzyme was determined by labeling with bromo-[3H]conduritol B 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.
|Translated title of the contribution||Intracellular activity of lysosomal glucosylceramidase measured with 4-nonylumbelliferyi β-glucoside|
|Number of pages||10|
|Journal||Biological Chemistry Hoppe-Seyler|
|State||Published - 1989|
- Intracellular activity of glucosylceramidase
- transport kinetics
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