Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells
Summary: GlcNAc-1-phosphotransferase (GNPT) is the key enzyme for tagging lysosomal hydrolases with the mannose 6-phosphate moiety for delivery to the lysosome. Here, we present the assay for measuring endogenous GNPT activity in SK-MEL-30 cells. We provide details for preparing the [3H]UDP-GlcNAc d...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725003417 |
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| Summary: | Summary: GlcNAc-1-phosphotransferase (GNPT) is the key enzyme for tagging lysosomal hydrolases with the mannose 6-phosphate moiety for delivery to the lysosome. Here, we present the assay for measuring endogenous GNPT activity in SK-MEL-30 cells. We provide details for preparing the [3H]UDP-GlcNAc donor substrate and conditions for the enzymatic transfer of [3H]GlcNAc-1-P to the methyl α-D-mannopyranoside acceptor (α-MM). We then describe the chromatography steps to specifically separate the [3H]GlcNAc-1-P-αMM GNPT reaction product from unreacted [3H]UDP-GlcNAc that is bound to the quaternary aminoethyl (QAE) Sephadex A-25 matrix.For complete details on the use and execution of this protocol, please refer to Doray et al.1 and Yang et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
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| ISSN: | 2666-1667 |