Selective Fluorogenic β-Glucocerebrosidase Substrates for Convenient Analysis of Enzyme Activity in Cell and Tissue Homogenates

Peer reviewed: 
Yes, item is peer reviewed.
Scholarly level: 
Faculty/Staff
Final version published as: 

Deen, M. C., Proceviat, C., Shan, X., Wu, L., Shen, D. L., Davies, G. J., & Vocadlo, D. J. (2020). Selective Fluorogenic β-Glucocerebrosidase Substrates for Convenient Analysis of Enzyme Activity in Cell and Tissue Homogenates. ACS Chemical Biology, 15(4), 824–829. https://doi.org/10.1021/acschembio.9b01044.

Date created: 
2020-02-28
Identifier: 
DOI: 10.1021/acschembio.9b01044
Keywords: 
Peptides and proteins
Central nervous system
Assays
Rodent models
Selectivity
Abstract: 

Within 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.

Description: 

The full text of this paper will be available in February, 2021 due to the embargo policies of Journal of the ACS Chemical Biology. Contact summit@sfu.ca to enquire if the full text of the accepted manuscript can be made available to you.

Language: 
English
Document type: 
Article
Rights: 
Rights remain with the authors.
Statistics: