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Protocols for Glycosyltransferase Assays: Ganglioside Globoside and Lewis-X Intermediate-Lactosylceramide Biosyntheses in Eukaryotic Systems

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Gangliosides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1804))

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Abstract

Protocols for assay of 24 different Glycolipid-Glycosyltransferases (GSL-GLTs) of the eukaryotic systems are described. Problems of quantitating the activities in crude membranes are also described. Different separation methods (for separation of substrate, donors, and the product of the reaction) have been described based on the paper chromatography or high voltage paper electrophoresis in 1.0% Na2B4O7. Liquid Scintillation counting system was used for quantitation of the enzymatic product. In the assay of each GSL-GLT it is recommended to compare the selected method to be used with the exact conditions used by the authors published previously. As a test case for these assays the following kinetic parameters for Lactosylceramide Synthase, GalT-2 (UDP-Gal: Glc-Cer β1-4-galactosyltransferase), (Km of glucosylceramide = 1.65 × 10−4 M; Km for UDP-Gal = 0.5 × 10−4 M; V max is determined in the presence of optimum detergent concentrations (2–15 mg/ml of Cutscum–Triton X-100, 2:1); Mn++ and Mg++, 10–20 mM) has been reported. The importance of use of GalT-2 assay method (as a model system) in the purified Golgi-rich membranes from 13-day-old embryonic chicken brains (13-ECB) is described.

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Acknowledgment

We thank Mrs. Dorisanne Nielsen and Mr. Eric Kuehner for their help during preparation of the manuscript. The Jacob Javits Research Award from NIH-NINDS NS-18005, Coleman Cancer Foundation, and NCI grant-CA-14764 to S. Basu; and a grant-in-aid from Siemens Corporation to M. Basu supported this research work.

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Authors and Affiliations

  1. Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, IN, USA

    Subhash Basu, Arun Agarwal & Manju Basu

  2. Cancer Drug Delivery Research Foundation, Notre Dame, IN, USA

    Subhash Basu, Arun Agarwal & Manju Basu

  3. Diagnostic Division, Siemens Corp, Shanghai, People’s Republic of China

    Rui Ma

  4. The Falk Ctr. for Molecular Therapeutics, Northwestern University, Evanston, IL, USA

    Joseph R. Moskal

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  1. Subhash Basu
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  2. Arun Agarwal
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  3. Manju Basu
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  4. Rui Ma
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  5. Joseph R. Moskal
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Corresponding author

Correspondence to Subhash Basu .

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Editors and Affiliations

  1. Department of Medical Biotech & Translational Medicine, University of Milan, Segrate, Milano, Italy

    Sandro Sonnino

  2. Department of Medical Biotech & Translational Medicine, University of Milan, Segrate, Milano, Italy

    Alessandro Prinetti

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Basu, S., Agarwal, A., Basu, M., Ma, R., Moskal, J.R. (2018). Protocols for Glycosyltransferase Assays: Ganglioside Globoside and Lewis-X Intermediate-Lactosylceramide Biosyntheses in Eukaryotic Systems. In: Sonnino, S., Prinetti, A. (eds) Gangliosides. Methods in Molecular Biology, vol 1804. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8552-4_8

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  • DOI: https://doi.org/10.1007/978-1-4939-8552-4_8

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