当前位置： SCI文献检索 > CLINICAL CHEMISTRY期刊下所有文献 > Specific determination of beta-galactocerebrosidase activity via competitive inhibition of beta-galactosidase.

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.

journal_name

Clin Chem

journal_title

Clinical chemistry

authors

Martino S,Tiribuzi R,Tortori A,Conti D,Visigalli I,Lattanzi A,Biffi A,Gritti A,Orlacchio A

doi

10.1373/clinchem.2008.115873

subject

Has Abstract

pub_date

2009-03-01 00:00:00

pages

541-8

issue

eissn

0009-9147

issn

1530-8561

pii

clinchem.2008.115873

journal_volume

pub_type

杂志文章

在线工具

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