An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes

E. Kohen, C. Kohen, J. G. Hirschberg, R. Santus, G. Grabowski, W. Mangel, S. Gatt, J. Prince

Research output: Contribution to journalArticle

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Abstract

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization 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 (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.

Original languageEnglish
Pages (from-to)167-177
Number of pages11
JournalCell Biochemistry and Function
Volume11
Issue number3
StatePublished - Jan 1 1993

Fingerprint

beta-Glucosidase
Fibroblasts
Apolipoproteins E
Lysosomes
Low Density Lipoprotein Receptor-Related Protein-1
Lysosomal Storage Diseases
Gene therapy
Cytotoxins
Enzyme activity
Golgi Apparatus
Enzymes
Bovine Serum Albumin
Endoplasmic Reticulum
Genetic Therapy
Detergents
Assays
Fluorescence
Ligands
Membranes
Lipids

Keywords

  • β-glucosidase
  • Fibroblasts
  • Fluorogenic probes
  • Gaucher's disease
  • Microspectrofluorimetry

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry

Cite this

Kohen, E., Kohen, C., Hirschberg, J. G., Santus, R., Grabowski, G., Mangel, W., ... Prince, J. (1993). An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes. Cell Biochemistry and Function, 11(3), 167-177.

An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes. / Kohen, E.; Kohen, C.; Hirschberg, J. G.; Santus, R.; Grabowski, G.; Mangel, W.; Gatt, S.; Prince, J.

In: Cell Biochemistry and Function, Vol. 11, No. 3, 01.01.1993, p. 167-177.

Research output: Contribution to journalArticle

Kohen, E, Kohen, C, Hirschberg, JG, Santus, R, Grabowski, G, Mangel, W, Gatt, S & Prince, J 1993, 'An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes', Cell Biochemistry and Function, vol. 11, no. 3, pp. 167-177.
Kohen E, Kohen C, Hirschberg JG, Santus R, Grabowski G, Mangel W et al. An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes. Cell Biochemistry and Function. 1993 Jan 1;11(3):167-177.
Kohen, E. ; Kohen, C. ; Hirschberg, J. G. ; Santus, R. ; Grabowski, G. ; Mangel, W. ; Gatt, S. ; Prince, J. / An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes. In: Cell Biochemistry and Function. 1993 ; Vol. 11, No. 3. pp. 167-177.
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