{"doi":"10.1016/j.xpro.2025.103935","title":"Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells","abstract":"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 [ 3 H]UDP-GlcNAc donor substrate and conditions for the enzymatic transfer of [ 3 H]GlcNAc-1-P to the methyl α-D-mannopyranoside acceptor (α-MM). We then describe the chromatography steps to specifically separate the [ 3 H]GlcNAc-1-P-αMM GNPT reaction product from unreacted [ 3 H]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 • Steps for measuring endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells • Instructions for preparing [ 3 H]UDP-GlcNAc donor substrate for radiometric assay • Guidance on performing chromatography separation and quantifying reaction products Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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 [ 3 H]UDP-GlcNAc donor substrate and conditions for the enzymatic transfer of [ 3 H]GlcNAc-1-P to the methyl α-D-mannopyranoside acceptor (α-MM). We then describe the chromatography steps to specifically separate the [ 3 H]GlcNAc-1-P-αMM GNPT reaction product from unreacted [ 3 H]UDP-GlcNAc that is bound to the quaternary aminoethyl (QAE) Sephadex A-25 matrix.","journal":"STAR Protocols","year":2025,"id":545293,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9537,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1154191,"name":"Danielle Henn","orcid":null,"position":1,"is_corresponding":false},{"id":1256682,"name":"Yang Xi","orcid":"0000-0002-0648-1728","position":2,"is_corresponding":false},{"id":486997,"name":"Ming Li","orcid":"0000-0001-6291-9328","position":3,"is_corresponding":false},{"id":471360,"name":"Patricia Dickson","orcid":"0000-0003-3467-8488","position":4,"is_corresponding":false},{"id":691057,"name":"Balraj Doray","orcid":"0000-0003-3347-5013","position":0,"is_corresponding":true}],"reference_count":10,"raw_metadata":null,"created_at":"2026-07-19T02:53:23.001995Z","pmid":"40638380","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}