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Specific determination of beta-galactocerebrosidase activity via competitive inhibition of beta-galactosidase.

AbstractBACKGROUND:
The determination of cellular beta-galactocerebrosidase activity is an established procedure to diagnose Krabbe disease and monitor the efficacy of gene/stem cell-based therapeutic approaches aimed at restoring defective enzymatic activity in patients or disease models. Current biochemical assays for beta-galactocerebrosidase show high specificity but generally require large protein amounts from scanty sources such as hematopoietic or neural stem cells. We developed a novel assay based on the hypothesis that specific measurements of beta-galactocerebrosidase activity can be performed following complete inhibition of beta-galactosidase activity.
METHODS:
We performed the assay using 2-7.5 microg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-beta-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO(3). Reactions were incubated for 30 min at 37 degrees C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (lambda(ex) 360 nm, lambda(em) 446 nm).
RESULTS:
AgNO(3) was a competitive inhibitor of beta-galactosidase [inhibition constant (K(i)) = 0.12 micromol/L] and completely inhibited beta-galactosidase activity when used at a concentration of 11 micromol/L. Under this condition, the beta-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect beta-galactocerebrosidase activity in as little as 2 microg cell protein extract or 7.5 microg tissue. Assay validation was performed using (a) brain tissues from wild-type and twitcher mice and (b) murine GALC(-/-) hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors.
CONCLUSIONS:
The procedure is straightforward, rapid, and reproducible. Within a clinical context, our method unequivocally discriminated cells from healthy subjects and Krabbe patients and is therefore suitable for diagnostic applications.
AuthorsSabata Martino, Roberto Tiribuzi, Andrea Tortori, Daniele Conti, Ilaria Visigalli, Annalisa Lattanzi, Alessandra Biffi, Angela Gritti, Aldo Orlacchio
JournalClinical chemistry (Clin Chem) Vol. 55 Issue 3 Pg. 541-8 (Mar 2009) ISSN: 1530-8561 [Electronic] United States
PMID19147730 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Silver Nitrate
  • beta-Galactosidase
  • Galactosylceramidase
Topics
  • Animals
  • Cells, Cultured
  • Chromatography, Gel
  • Enzyme Activation (drug effects)
  • Galactosylceramidase (analysis, metabolism)
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Silver Nitrate (pharmacology)
  • Tissue Culture Techniques
  • beta-Galactosidase (antagonists & inhibitors, metabolism)

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