Pitfalls in the use of artificial substrates for the diagnosis of Gaucher's disease.

Ben‐Yoseph, Yoav; Nadler, Henry L.

doi:10.1136/jcp.31.11.1091

Cited by 19 publications

(7 citation statements)

References 8 publications

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“…Fluorescence-based GBA Activity Assays-The activity of cellular GBAs was analyzed using 4-methylumbelliferyl-␤-D-glucopyranoside (4-MU-␤-D-glucopyranoside) (Sigma) (26,27) as a fluorescent substrate. Cleavage of 4-MU-␤-D-glucopyranoside was monitored in real time in a Fluostar Omega reader (BMG Labtech) at 29°C using the filter pair 355 nm/460 nm for excitation and emission, respectively.…”

Section: Methodsmentioning

confidence: 99%

The Non-lysosomal β-Glucosidase GBA2 Is a Non-integral Membrane-associated Protein at the Endoplasmic Reticulum (ER) and Golgi

Körschen

Yildiz

Raju

et al. 2013

Journal of Biological Chemistry

101

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Background:The ␤-glucosidase GBA2 degrades glucosylceramide (GlcCer) outside the lysosomes. Results: GBA2 is not an integral membrane protein but rather is membrane-associated at the ER and Golgi. Conclusion: GBA2 is located in a key position for a lysosome-independent route of GlcCer-dependent signaling. Significance: Understanding the localization and enzymatic properties of GBA2 is crucial for investigating the role of nonlysosomal glucosylceramide in Gaucher disease pathology.

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Section: Methodsmentioning

confidence: 99%

The Non-lysosomal β-Glucosidase GBA2 Is a Non-integral Membrane-associated Protein at the Endoplasmic Reticulum (ER) and Golgi

Körschen

Yildiz

Raju

et al. 2013

Journal of Biological Chemistry

101

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“…Although these fluorogenic substrates are sensitive in the assay and easy to use, they may not reflect physiological conditions in cells. In addition, the specificity of fluorogenic substrates could be a problem in assays using cell or tissue preparations instead of purified recombinant enzymes, as they may be cleaved by other cellular enzymes present in these crude preparations [16–18]. Alternatively, the natural substrate, glucosylceramide, can be used in the glucocerebrosidase assay to more accurately measure glucocerebrosidase activity in enzyme preparations from cells and tissues.…”

Section: Introductionmentioning

confidence: 99%

A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

et al. 2011

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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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“…Recently, it was reported that the various fluorometric diagnostic assays give ambiguous results when liver is the source of the enzyme [3,9]. The problem with using a fluorometric assay is that liver is a particularly rich source of the non-specific P-glucosidase.…”

Section: Introductionmentioning

confidence: 99%

A revised fluorometric assay for Gaucher's disease using conduritol-β-epoxide with liver as the source of β-glucosidase

Daniels

Glew

Radin

et al. 1980

Clinica Chimica Acta

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To date, enzymatic diagnosis of Gaucher's disease via a fluorometric assay procedure which utilizes 4-methylumbelliferyl-beta-D-glucopyranoside as a substrate has not been possible when liver serves as the source of enzyme since currently employed fluorometric procedures cannot adequately differentiate between a broad-specificity beta-glucosidase and lysosomal glucocerebrosidase activities in crude extracts of liver. Incorporation of conduritol-beta-epoxide into the incubation medium for the fluorometric assay allows one to selectively measure the glucocerebrosidase activity present in a given liver extract. In five cases of Gaucher's disease this revised fluorometric procedure proved as effective as the assy procedure which utilizes authentic, radiolabeled glucocerebroside as the substrate in demonstrating a deficiency of glucocerebrosidase activity in liver.

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Pitfalls in the use of artificial substrates for the diagnosis of Gaucher's disease.

Cited by 19 publications

References 8 publications

The Non-lysosomal β-Glucosidase GBA2 Is a Non-integral Membrane-associated Protein at the Endoplasmic Reticulum (ER) and Golgi

The Non-lysosomal β-Glucosidase GBA2 Is a Non-integral Membrane-associated Protein at the Endoplasmic Reticulum (ER) and Golgi

A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

A revised fluorometric assay for Gaucher's disease using conduritol-β-epoxide with liver as the source of β-glucosidase

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