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Selective Fluorogenic β‑Glucocerebrosidase Substrates for Convenient Analysis of Enzyme Activity in Cell and Tissue Homogenates
journal contribution
posted on 2020-03-16, 13:39 authored by Matthew C. Deen, Cameron Proceviat, Xiaoyang Shan, Liang Wu, David L. Shen, Gideon J. Davies, David J. VocadloWithin
mammals, there are often several functionally related glycoside hydrolases,
which makes monitoring their activities problematic. This problem
is particularly acute for the enzyme β-glucocerebrosidase (GCase),
the malfunction of which is a key driver of Gaucher’s disease
(GD) and a major risk factor for Parkinson’s disease (PD).
Humans harbor two other functionally related β-glucosidases
known as GBA2 and GBA3, and the currently used fluorogenic substrates
are not selective, which has driven the use of complicated subtractive
assays involving the use of detergents and inhibitors. Here we describe
the preparation of fluorogenic substrates based on the widely used
nonselective substrate resorufin β-d-glucopyranoside.
Using recombinant enzymes, we show that these substrates are highly
selective for GCase. We also demonstrate their value through the analysis
of GCase activity in brain tissue homogenates from transgenic mice
expressing mutant human GCase and patient fibroblasts expressing mutant
GCase. This approach simplifies the analysis of cell and tissue homogenates
and should facilitate the analysis of clinical and laboratory tissues
and samples.