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

Abstract

BACKGROUND:

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.

• Authors: Sabata Martino, Roberto Tiribuzi, Andrea Tortori, Daniele Conti, Ilaria Visigalli, Annalisa Lattanzi, Alessandra Biffi, Angela Gritti, Aldo Orlacchio
• Journal: Clinical chemistry (Clin Chem) Vol. 55 Issue 3 Pg. 541-8 (Mar 2009) ISSN: 1530-8561 [Electronic] England
• PMID: 19147730 (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)