Specific Determination of β-Galactocerebrosidase Activity via Competitive Inhibition of β-Galactosidase

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 55; no. 3; pp. 541 - 548
• Main Authors: Martino, Sabata, Tiribuzi, Roberto, Tortori, Andrea, Conti, Daniele, Visigalli, Ilaria, Lattanzi, Annalisa, Biffi, Alessandra, Gritti, Angela, Orlacchio, Aldo
• Format: Journal Article
• Language: English
• Published: Washington, DC American Association for Clinical Chemistry 01.03.2009
• Subjects: Analytical, structural and metabolic biochemistry; Animals; beta-Galactosidase - antagonists & inhibitors; beta-Galactosidase - metabolism; Biological and medical sciences; Cells, Cultured; Chromatography, Gel; Enzyme Activation - drug effects; Fundamental and applied biological sciences. Psychology; Galactosylceramidase - analysis; Galactosylceramidase - metabolism; Humans; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Mice; Mice, Inbred C57BL; Silver Nitrate - pharmacology; Tissue Culture Techniques; Glycosylases; Specific activity; Galactosylceramidase; Enzyme; Glycosidases; Clinical biology; Hydrolases; Biochemistry; Molecular biology; Competitive inhibition; β-Galactosidase
• ISSN: 0009-9147; 1530-8561; 1530-8561
• DOI: 10.1373/clinchem.2008.115873

Background: The determination of cellular β-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 β-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 β-galactocerebrosidase activity can be performed following complete inhibition of β-galactosidase activity. Methods: We performed the assay using 2–7.5 μg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-β-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO3. Reactions were incubated for 30 min at 37 °C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (λex 360 nm, λem 446 nm). Results: AgNO3 was a competitive inhibitor of β-galactosidase [inhibition constant (Ki) = 0.12 μmol/L] and completely inhibited β-galactosidase activity when used at a concentration of 11 μmol/L. Under this condition, the β-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect β-galactocerebrosidase activity in as little as 2 μg cell protein extract or 7.5 μg 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.