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

Abstract

BACKGROUND:The determination of cellular ␤-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 ␤-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 ␤-galactocerebrosidase activity can be performed following complete inhibition of ␤-galactosidase activity.

Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy

“…ARSA activity was determined using the 4-methylumbelliferyl-sulfate substrate and calculated by subtracting the value obtained in the presence of AgNO 3 [Arylsulphatase B (ARSB) activity] from that measured in the absence of the inhibitor (ARSA + ARSB activity). Both assays were performed according to previously described protocols (64,65) that have been validated in our previous studies (15,19). …”

Original article Diagnostic difficulties in Krabbe disease: a report of two cases and review of literature

“…It can be speculated that during the long incubation time (18 hr) other, not substrate-specific enzymes, present in the homogenate, can be involved in the degradation of the artificial substrate. Such an enzyme may be lysosomal β-galactosidase and its undesirable action should be specifically inhibited [17]. It should be also emphasized that enzyme activities measured in cultured skin fibroblasts are relevant to higher variability than in isolated blood leukocytes of the examined individual as the culture conditions are very changeable.…”

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

“…To distinguish the two enzyme activities we followed the protocol described by Martino et al which employs AgNO3 as a selective competitive inhibitor of lysosomal acid β-galactosidase. [38] Preincubation of brain and kidney lysates with 10 µM of probe 3 resulted in significant decrease of GALC activity (approximately 50% of untreated values) in these tissues, while β-galactosidase activity was unaltered ( Figure 3C and D). In contrast, incubation of the lysates with 0.5 µM of inhibitor 1 completely abrogated both enzyme activities.…”

“…For measurement of β-galactosidase and β-galactocerebrosidase activity we followed the protocol previously described by Martino et al [38] with slight alterations. Briefly, a volume of tissue lysate equivalent to 7.5 µg of protein was completed with water (6.25 µL total volume) and exposed to the indicated concentration of ABP 3 or inhibitor 1 (6.25 µL 2x solution in McIlvaine buffer pH 4.3) for 60 min at 37°C.…”