D mannose 6 phosphate

Manufactured by Merck Group

D-mannose 6-phosphate is a lab equipment product. It is a sugar phosphate that serves as a key intermediate in carbohydrate metabolism.

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3 protocols using d mannose 6 phosphate

Inhibition of Enzyme Uptake in Brain Cells

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Uptake inhibition assays were performed in MPS IIIB fibroblasts and two brain tumor-derived cell lines, Dao Y and Es (cerebellar medulloblastoma and glioblastoma, respectively, kindly provided by Dr. J Lasky, Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CA). Cells were seeded in 12-well plates and grown to confluence. Culture medium was replaced with 0.5 ml of EMEM without serum prior to the addition of uptake inhibitors. 5 µg/ml recombinant human IGF-II (R&D Systems; Minneapolis, MN) or 5 mM D-mannose 6-phosphate (Sigma-Aldrich; St. Louis, MO) was applied to the cells for 10 minutes prior to applying purified rhNAGLU-IGF-II at a final concentration of 160 units/ml. Following 4 hours incubation at 37°C and 5% CO₂, cells were harvested and intracellular NAGLU activity was measured as described above. The enzyme activity measured in MPS IIIB fibroblasts with rhNAGLU-IGF-II treatment alone was defined as 100% and the intracellular enzyme activity observed in the presence of inhibitor(s) was normalized to rhNAGLU-IGF-II activity. The endogenous intracellular enzyme activity measured in brain tumor-derived cell lines was defined as 100% and the enzyme uptake with or without inhibitor was normalized to this value.

Kan S.H., Troitskaya L.A., Sinow C.S., Haitz K., Todd A.K., Di Stefano A., Le S.Q., Dickson P.I, & Tippin B.L. (2014). Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts. The Biochemical journal, 458(2), 281-289.

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Purification and Characterization of QDK-PMM1

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DEAE-Sepharose ff, Superdex-75, Butyl Sepharose ff, were purchased from GE Healthcare Life Sciences. Phosphoglucose isomerase from rabbit muscle, phosphomannose isomerase from E. coli, glucose 6-phosphate dehydrogenase from baker's yeast (S. cerevisiae), phosphoglucomutase from rabbit muscle, α-D-Glucose 1,6-bisphosphate potassium salt hydrate, α-D-Glucose 1-phosphate disodium salt hydrate, α-D(+)Mannose 1-phosphate sodium salt hydrate, D-Glucose 6-phosphate, D-Mannose 6-phosphate, β-Nicotinamide adenine dinucleotide phosphate sodium salt, were purchased from Sigma-Aldrich. Sypro Orange was from Invitrogen Molecular Probes. Sodium orthovanadate was from Acros Organics. Creatine phosphate disodium salt tetrahydrate was from Alfa Aesar. Clodronate ((Dichloro-phosphono-methyl)phosphonic acid) and neridronate ((6-Amino-1-hydroxyhexane-1,1-diyl)bis(phosphonic acid) were from ABIOGEN-PHARMA.
Mannose 1,6-bisphosphate was synthesized as described [18 (link)] and purified on an AG1x8 hydroxide form column by running a step gradient 0–1 M NaCl. Fractions were analyzed by ¹H- and ³¹P-NMR spectroscopy. An internal standard (Trimethylsilylpropanoic acid) was used in order to measure the concentration. All the other reagents were of analytical grade. The ORF encoding QDK-PMM1 was obtained by de novo gene synthesis and was purchased by GeneCust, Luxemburg; it is available upon request.

Citro V., Cimmaruta C., Liguori L., Viscido G., Cubellis M.V, & Andreotti G. (2017). A mutant of phosphomannomutase1 retains full enzymatic activity, but is not activated by IMP: Possible implications for the disease PMM2-CDG. PLoS ONE, 12(12), e0189629.

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Metabolite Extraction and Derivatization

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D-Erythrose 4-phosphate (E4P), glycolaldehyde, D-glucose 6-phosphate, D-mannose 6-phosphate, methoxyamine hydrochloride, pyridine and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). D-Allose and D-altrose were purchased from Macklin Biochemical (Shanghai, China). Other regents such as D-glucose, D-mannose, ATP, ADP, thiamine pyrophosphate (TPP), and MgCl₂ were all purchased from Sangon Biotech (Shanghai, China), unless noted otherwise. Escherichia coli DH5α was used for plasmid construction and preservation. Escherichia coli BL21 (DE3) was used for protein expression. Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl) was used for E. coli cell culture and recombinant protein expression, supplemented with 50 μg/ml kanamycin or 100 μg/ml ampicillin when necessary.

Mao Y., Yuan Q., Yang X., Liu P., Cheng Y., Luo J., Liu H., Yao Y., Sun H., Cai T, & Ma H. (2021). Non-natural Aldol Reactions Enable the Design and Construction of Novel One-Carbon Assimilation Pathways in vitro. Frontiers in Microbiology, 12, 677596.

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