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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| Language: | English |
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Elsevier
2025-09-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725003417 |
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| author | Balraj Doray Danielle Henn Xi Yang Ming Li Patricia Dickson |
| author_facet | Balraj Doray Danielle Henn Xi Yang Ming Li Patricia Dickson |
| author_sort | Balraj Doray |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-69c528ae8a8a4341938b2c5ffb87c7af |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-69c528ae8a8a4341938b2c5ffb87c7af2025-08-20T03:33:38ZengElsevierSTAR Protocols2666-16672025-09-016310393510.1016/j.xpro.2025.103935Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cellsBalraj Doray0Danielle Henn1Xi Yang2Ming Li3Patricia Dickson4Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA; Corresponding authorDepartment of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USADepartment of Biological Sciences, University of Denver, Denver, CO 80208, USADepartment of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USADepartment of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA; Corresponding authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2666166725003417Cell BiologyMolecular BiologyProtein Biochemistry |
| spellingShingle | Balraj Doray Danielle Henn Xi Yang Ming Li Patricia Dickson Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells STAR Protocols Cell Biology Molecular Biology Protein Biochemistry |
| title | Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells |
| title_full | Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells |
| title_fullStr | Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells |
| title_full_unstemmed | Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells |
| title_short | Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells |
| title_sort | protocol to measure endogenous glcnac 1 phosphotransferase activity in sk mel 30 cells |
| topic | Cell Biology Molecular Biology Protein Biochemistry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725003417 |
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