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A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

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Abstract

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.

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Acknowledgments

This research was supported by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research and the Intramural Research Programs of the National Human Genome Research Institute and the National Institutes of Health.

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Authors and Affiliations

  1. NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892-3370, USA

    Omid Motabar, William Leister, Ke Liu, Noel Southall, Wenwei Huang, Juan J. Marugan & Wei Zheng

  2. Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892-3708, USA

    Omid Motabar, Ehud Goldin & Ellen Sidransky

Authors
  1. Omid Motabar
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  2. Ehud Goldin
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  3. William Leister
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  4. Ke Liu
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  5. Noel Southall
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  6. Wenwei Huang
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  7. Juan J. Marugan
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  8. Ellen Sidransky
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  9. Wei Zheng
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Correspondence to Wei Zheng.

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Motabar, O., Goldin, E., Leister, W. et al. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide. Anal Bioanal Chem 402, 731–739 (2012). https://doi.org/10.1007/s00216-011-5496-z

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  • DOI: https://doi.org/10.1007/s00216-011-5496-z

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