AN IN-SITU STUDY OF BETA-GLUCOSIDASE ACTIVITY IN NORMAL AND GAUCHER FIBROBLASTS WITH FLUOROGENIC PROBES

Beta-glucosidase activity was evaluated in situ by means of fluorogeni c probes in normal human fibroblasts and fibroblasts from homozygous c arriers of the Gaucher trait. Probe internalization, targeting to lyso somes and post-cleavage probe retention were the primary concerns. Int ernalization and targeting were attempted by in situ photosensitized l abilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bi s) beta-glucopyranoside was appreciably above the rather large pre-cle avage emission. In cells incubated overnight with nonylumbelliferylbet aglucoside (UG9) in the presence of bovine serum albumin and in the ab sence of ApoE, the probe was dealt with as a cytotoxic agent, accumula ting in a paranuclear cap, most likely comprising elements of the endo plasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysoso mes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibi ted weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-gl ucosidase activity. Cleavage of UG9 was nearly totally suppressed in G aucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts w as evident but to a lesser degree. The in situ method of fluorogenic a ssay established for beta-glucosidase deficiency, is in principle appl icable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.