title: 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.affiliation: Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia Molecolare, University of Perugia, Perugia, Italy.
authors.forename: Sabata
authors.initials: S
authors.lastname: Martino
authors.forename: Roberto
authors.initials: R
authors.lastname: Tiribuzi
authors.forename: Andrea
authors.initials: A
authors.lastname: Tortori
authors.forename: Daniele
authors.initials: D
authors.lastname: Conti
authors.forename: Ilaria
authors.initials: I
authors.lastname: Visigalli
authors.forename: Annalisa
authors.initials: A
authors.lastname: Lattanzi
authors.forename: Alessandra
authors.initials: A
authors.lastname: Biffi
authors.forename: Angela
authors.initials: A
authors.lastname: Gritti
authors.forename: Aldo
authors.initials: A
authors.lastname: Orlacchio
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D005698:Galactosylceramidase / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D012835:Silver Nitrate / Q000494:pharmacology; D046509:Tissue Culture Techniques; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D012835:Silver Nitrate; D001616:beta-Galactosidase; D005698:Galactosylceramidase